Bispecific single chain Fv antibody molecules and methods of use therof

ABSTRACT

Bispecific single chain antibody molecules are disclosed which may be used to advantage to treat various forms of cancer associated with the overexpression of members of the EGFR protein family.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of U.S. Ser. No. 11/943,367, filed onNov. 20, 2007, now U.S. Pat. No. 8,329,873, which is a Divisional ofU.S. Ser. No. 11/154,103, filed on Jun. 15, 2005, now U.S. Pat. No.7,332,585, which is a continuation-in-part of U.S. Ser. No. 10/406,830,filed on Apr. 4, 2003, now U.S. Pat. No. 7,332,580 which claims priorityand benefit of U.S. Provisional Application U.S. Ser. No. 60/370,276,filed on Apr. 5, 2002, all of which are incorporated herein by referencein their entirety for all purposes.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

This invention was made with government support under grant no. CA06927awarded by the National Institutes of Health (NCI) and grant no.DAMD17-01-1-0520 awarded by the United States Army Medical Research andMateriel Command. The government has certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates to the fields of immunology and oncology,and more specifically, to bispecific antibody molecules (e.g. bs scFv)that can be used to advantage in the detection and/or treatment ofvarious cancers that overexpress the Epidermal Growth Factor Receptor(EGFR) family of proteins. Certain illustrative bispecific scFv antibodymolecules of the invention have binding specificities for either twodistinct epitopes of a single member of the EGFR family or alternativelyspecificity for two distinct members of the EGFR family.

BACKGROUND OF THE INVENTION

The Epidermal Growth Factor Receptor (EGFR) signaling pathway plays animportant role in the development and spread of cancer throughout thebody. EGFR, also known as erb-b1, is a member of a family of four genesthat also includes HER2/neu (erb-b2), HER3 (erb-b3) and HER4 (erb-b4).EGFR is expressed in a wide range of solid tumors, including coloncancers, head and neck cancers, pancreatic cancers, ovarian cancers, andbreast cancers.

HER2/neu is a cell surface receptor protein with tyrosine kinaseactivity. The complete protein consists of three parts: an intracellularcytoplasmic domain, a short hydrophobic transmembrane segment and anextracellular domain (ECD) that is responsible for ligand binding. Thisreceptor protein is expressed on the cell membrane of a variety ofepithelial cell types and, through binding of specific growth factors,regulates various aspects of cell growth division.

Her2/neu, the gene that encodes for the HER2/neu protein, is a member ofa group of genes known as proto-oncogenes. Proto-oncogenes encodeimportant proteins, such as growth factors, growth factor receptors, andapoptotic proteins, that are involved in normal cell growth anddifferentiation. When proto-oncogenes are altered by point mutation,translocation or gene amplification, they produce growth signals thatmay lead to aberrant cellular transformation and the development ofcancer.

While Her2/neu can be expressed at low levels in many normal cells, itis typically overexpressed in a variety of cancers. Overexpression ofHer2/neu is caused in most cases by an increase in copy number of thegene (gene amplification) and/or by an increase in expression level ofthe Her2/neu genes in the cell. Overexpression of this growth factorreceptor plays a key role in tumor progression by causing a higher rateof cell growth and oncogenic transformation. Gene amplification of theHer2/neu gene has been observed in a variety of cancer types, including,breast, ovarian, endometrial, gastric, pancreatic, prostate and salivarygland (Hynes and Stern (1994) Biochim Biophys Acta., 1198: 165-184). Inbreast cancer patients, HER2/neu has also been shown to be of clinicalimportance as it is associated with poor prognosis, tumor recurrence andshortened survival in breast cancer patients (Seshadri et al. (1993) J.Clin. Oncol., 11: 1936-1942; Berger et al. (1988) Cancer Res., 48:1238-1243; O'Reilly et al. (1991) Br. J. Cancer, 63: 444-446).

Currently, a great deal of attention has focused on the development ofnovel immunotherapy strategies for the treatment of cancer. One suchstrategy is antibody-based cancer therapy. A major goal ofantibody-based cancer therapy is to specifically deliver toxic payloadssuch as radioisotopes, toxins or drugs to tumors. The size range ofantibody binding site-based molecules includes: IgM (1000 kDa), IgG (150kDa), F(ab′)₂ (100 kDa), Fab (50 kDa), (scFv′)₂ (55 kDa) and scFv (25kDa). In immunodeficient mice, larger molecules such as IgG and F(ab′)₂fragments are retained at high levels in human tumor xenografts with alow degree of specificity (Adams et al. (1992) Antibody, Immunoconj.Radiopharm., 5: 81-95; Milenic et al. (1991) J. Cancer Res. 51:6363-6371), while smaller molecules such as scFv, (scFv′)₂ and Fab areretained in tumors at comparatively lower levels with greatly improvedspecificity (Milenic et al. (1991) J. Cancer Res. 51: 6363-6371; Adamset al. (1993) Cancer Res. 53: 4026-4034; Beaumier et al. (1985) J. Nucl.Med. 26: 1172-1179; Colcher et al. (1990) J. Natl. Cancer Inst. 82:1191-1197).

The most prominent determinant of the above targeting properties is thesize of the antibody-based molecule relative to the renal threshold forfirst pass clearance. Another important feature of antibody-basedmolecules is valence, as significantly greater tumor retention has beenassociated with multivalent binding to target antigen (Milenic et al.(1991) J. Cancer Res. 51: 6363-6371; Adams et al. (1993) Cancer Res. 53:4026-4034; Adams et al. (1996) Proc. Amer. Assoc. Cancer Res. 37: 472;Wolf et al. (1993) Cancer Res. 53: 2560-2565).

Herceptin, a new form of immunotherapy targeting breast cancer, wasrecently developed to target cancer cells that overexpress Her2/neu.This treatment has been shown in clinical trials to provide effectivetreatment for patients with HER2/neu positive metastatic breast cancer.However, this drug treatment is costly and is associated withsignificant morbidity and mortality.

Several other types of therapy have been shown to be more or lesseffective in breast cancer patients whose tumors express elevated levelsof Her2/neu. These include, anthracycline therapy which is thought to bemore effective in patients with amplified Her2/neu expression, andhormonal therapy which is less effective in patients whose level ofHer2/neu expression is high.

Attention has also focused upon the generation of bivalent single chainFv-based antibody molecules with molecular weights in the range of therenal threshold for first pass clearance. These include 50 kDa diabodies(Holliger et al. (1993) Proc. Natl. Acad. Sci. USA, 90: 6444-6448), 55kDa (scFv′)₂ (Adams et al. (1993) Cancer Res. 53: 4026-4034), 60-65 kDaamphipathic helix-based scFv dimers (Pack et al. (1993) Bio/Technology11: 1271-1277; Pack (1992) Biochemistry 31: 1579-1584), and 80 kDa(scFv-C_(H)3)₂ LD minibodies and Flex minibodies (Hu et al. (1996)Cancer Res. 56: 3055-3061). While each of these proteins is capable ofbinding two antigen molecules, they differ in the orientation,flexibility and the span of their binding sites. It is believed thatthese new and innovative immunotherapies will help improve outcomes inbreast and other cancers which too frequently recur or progress despiteaggressive multi-modality therapy.

SUMMARY OF THE INVENTION

This invention pertains to the identification of bispecific (orpolyspecific) antibody molecules (e.g. bs scFv) that can be used toadvantage in the detection and/or treatment of various cancers thatoverexpress the Epidermal Growth Factor Receptor (EGFR) family ofproteins. Thus, in one embodiment this invention provides a bispecificantibody comprising an first antibody and a second antibody joined(directly or through a linker) to each other where the first antibodyand the second antibody bind specifically to different epitopes and thefirst antibody has binding specificity for (specifically binds) at leastone epitope on a member of the Epidermal Growth Factor Receptor proteinfamily, (e.g., EGFR, HER2/neu, HER3, HER4), and the second antibody hasbinding specificity for (specifically binds) a second epitope on amember of the Epidermal Growth Factor Receptor protein family which isdifferent from the first epitope is an epitope on a protein selectedfrom the group consisting of EGFR, HER2/neu, HER3 and HER4. In certainembodiments, the antibodies are joined by a linker, more preferably by apeptide linker, and most preferably by a peptide linker that lacks aproteolytic cleavage site (e.g., a linker having the amino acid sequenceof SEQ ID NO:37). In certain embodiments, the first and/or the secondantibody specifically binds an epitope specifically bound by an antibodyselected from the group consisting of C6.5, C6ML3-9, C6 MH3-B1, C6-B1D2,F5, HER3.A5, HER3.F4, HER3.H1, HER3.H3, HER3.E12, HER3.B12, EGFR.E12,EGFR.C10, EGFR.B11, EGFR.E8, HER4.B4, HER4.G4, HER4.F4, HER4.A8,HER4.B6, HER4.D4, HER4.D7, HER4.D11, HER4.D12, HER4.E3, HER4.E7, HER4.F8and HER4.C7. In certain embodiments, the first and/or the secondantibody comprise one, two, or all complementarity determining region(s)of an antibody selected from the group consisting of C6.5, C6ML3-9, C6MH3-B1, C6-B1D2, F5, HER3.A5, HER3.F4, HER3.H1, HER3.H3, HER3.E12,HER3.B12, EGFR.E12, EGFR.C10, EGFR.B11, EGFR.E8, HER4.B4, HER4.G4,HER4.F4, HER4.A8, HER4.B6, HER4.D4, HER4.D7, HER4.D11, HER4.D12,HER4.E3, HER4.E7, HER4.F8 and HER4.C7. In certain embodiments, thebispecific antibody or polyspecific antibody is encoded by a vectorcomprising a nucleic acid that encodes a polypeptide sequence selectedfrom the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3,SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8,SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 14, SEQ ID NO:20, and SEQ IDNO:22. In certain embodiments, the bispecific or polyspecific antibodyis encoded by a vector comprising two nucleic acid sequences encodingpolypeptides encoded by two nucleic acid sequences as described herein,e.g., independently selected from the group consisting of SEQ ID NO: 1,SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6,SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO:20,and SEQ ID NO:22. In certain instances, the bispecific antibody isencoded by a vector comprising, in certain instances, at least one, andin certain instances at least two nucleic acid nucleic acid sequence asdescribed herein, e.g., selected from the group consisting of SEQ ID NO:1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6,SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 14,SEQ ID NO:20, and SEQ ID NO:22. In certain instances the vector furthercomprises a nucleic acid sequence encoding a polypeptide having thesequence of SEQ ID NO:37.

In certain embodiments, the first and/or second antibodies (e.g. theantibodies described above) are single-chain antibodies (e.g. sc Fvantibodies). Where both the first and second antibodies are both singlechain antibodies, the antibodies are preferably directly attached (toform a single polypeptide) or attached through a linker, more preferablythrough a peptide linker (e.g. a peptide linker lacking a proteolyticcleavage site) to form a single chain bispecific or polyspecificantibody (e.g. bs-scFv). bispecific (or polyspecific) antibody is asingle chain antibody and said second antibody is a single chainantibody and said first antibody is coupled to said second antibody by apeptide linker.

In another embodiment, this invention includes a composition comprisinga bispecific or polyspecific antibody as disclosed and/or claimed hereinand a pharmaceutically acceptable carrier.

This invention also provides a method for treating cancer (e.g.mitigating one or more symptoms of cancer). The method typicallyinvolves administering to a patient (human or non-human animal) in needthereof a therapeutically effective amount of a bispecific orpolyspecific antibody as disclosed and/or claimed herein and apharmaceutically acceptable carrier. The cancer can include, but is notlimited to a cancer is selected from the group consisting of breast,colon, ovarian, endometrial, gastric, pancreatic, prostate and salivarygland cancer. The administration can be by any of a variety ofconvenient methods including systemic injectable administration,injection into a tumor or cancerous tissue, oral administration, and thelike.

In still another embodiment, this invention provides a method fortreating cancer (e.g. mitigating one or more symptoms of cancer). Themethod typically involves administering to a patient (human or non-humananimal) in need thereof a therapeutically effective amount of abispecific or polyspecific antibody as disclosed and/or claimed hereinand a pharmaceutically acceptable carrier, in combination with an othercytotoxic agent selected from the group consisting of a chemotherapeuticagent, external beam radiation, a targeted radioisotope, and a signaltransduction inhibitor. The cancer can include, but is not limited to acancer is selected from the group consisting of breast, colon, ovarian,endometrial, gastric, pancreatic, prostate and salivary gland cancer.The administration can be by any of a variety of convenient methodsincluding systemic injectable administration, injection into a tumor orcancerous tissue, oral administration, and the like.

In yet another embodiment, this invention provides a chimeric moietycomprising of a bispecific or polyspecific antibody as disclosed and/orclaimed herein coupled to an effector. Preferred effectors include, butare not limited to a cytotoxin, a label, a radionuclide, a drug, aliposome, a ligand, and an antibody. In certain instances, where theeffector is a polypeptide, the chimeric moiety is a fusion protein,preferably a recombinantly expressed fusion protein.

This invention also provides a method of specifically delivering ortargeting an effector molecule to a cell bearing a receptor fromEpidermal Growth Factor Receptor protein family (e.g., EGFR, HER2/neu,HER3 HER4). The method involves providing a chimeric moiety as describedand/or claimed herein, and contacting the cell with the chimeric moiety,whereby the chimeric moiety specifically binds to the cell. Preferredeffectors include, but are not limited to a cytotoxin, a label, aradionuclide, a drug, a liposome, a ligand, an antibody, etc. In certainembodiments, the chimeric moiety is a fusion protein. In certainembodiments, the cell is a cancer cell, preferably a cancer cell thatoverexpress one or more members of the EGFR protein family. Particularlypreferred cancer cells include, but are not limited to breast, colon,ovarian, endometrial, gastric, pancreatic, prostate and salivary glandcancer cells.

Also provided is a method of specifically killing and/or inhibiting thegrowth or proliferation of a cell bearing a receptor from EpidermalGrowth Factor Receptor protein family (e.g. EGFR, HER2/neu, HER3, HER4).The method typically involves providing a chimeric moiety as describedand/or claimed herein attached to a cytoxic or cytostatic effector (e.g.an a cytotoxin, a radioactive moiety, and a liposome comprising acytotoxic or cytostatic agent, and the like); and contacting said cellwith the chimeric moiety, whereby the chimeric moiety specifically bindsto the cell resulting in the death and/or inhibition of growth and/orproliferation of the cell. In certain embodiments, the chimeric moietyis a fusion protein. In certain embodiments, the cell is a cancer cell,preferably a cancer cell that overexpress one or more members of theEGFR protein family. Particularly preferred cancer cells include, butare not limited to breast, colon, ovarian, endometrial, gastric,pancreatic, prostate and salivary gland cancer cells.

This invention also provides methods of detecting and/or visualizingand/or diagnosing the presence of a cancer cell or tissue. The methodtypically involves contacting a cell or tissue with a chimeric moietycomprising a bispecific or polyspecific antibody as described hereinattached to a detectable label; and detecting the label where detectionof the label in association with the cell or tissue indicates thepresence of a cell or tissue expressing (or overexpressing one or moremembers of the Epidermal Growth Factor Receptor protein family.Preferred detectable labels include, but are not limited to a gammaemitter, a positron emitter, an MRI label, and a fluorescent orcolorimetric label. In certain instances, the detectable label is agamma emitter and the detecting comprises imaging with a gamma camera.In certain instances, the detectable label is a positron emitter and thedetecting comprises imaging with positron emission tomography (PET). Incertain instances, the detectable label is an MRI label and thedetecting comprises detecting with magnetic resonance imaging. Incertain embodiments, the cell or tissue expressing one or more membersof the Epidermal Growth Factor Receptor Protein family is a cell ortissue that overexpresses a protein selected from the group consistingof EGFR, HER2/neu, HER3 and HER4. The cell or tissue expressing one ormore members of the Epidermal Growth Factor Receptor Protein family is acan be a cancer cell or tissue (e.g., breast, colon, ovarian,endometrial, gastric, pancreatic, prostate, or salivary gland cancer).It is noted that the diagnostic assay can be a component of adifferential diagnosis of a cancer and/or can be used to type a canceras one that overexpresses one or members of the EGFR protein familyand/or the assay can be used to visualize a known cancer. In these (andother) instances, the assay need not be dispositive of the presence of acancer cell, but simply indicative of the likely presence of such a cellor tissue. In certain embodiments, the detecting comprises anon-invasive imaging technique. In certain embodiments, the detectingcomprises immunohistochemistry. In certain embodiments, the detectingcomprises detecting in a tissue sample or biopsy. In certainembodiments, the detecting comprises detecting in a tissue section. Incertain embodiments, the detecting is in vivo detection.

In accordance with the present invention, in certain embodiments, novelbispecific single chain Fv antibody molecules (bs-scFv) having bindingaffinity for members of the EGFR protein family are provided.

In certain preferred embodiments of the invention, the bs-scFvantibodies have a first and second arm that have binding affinity fortwo distinct epitopes on different members of the EGFR protein family(e.g., EGFR, HER2/neu, HER3 and HER4) or for two distinct epitopes on asingle member of the EGFR protein family, and are operably linked via anovel linker molecule which lacks proteolytic cleavage sites. Thislinker constitutes an aspect of the present invention. The arms that arepaired together to form the bs-scFv antibodies may be any one of thefollowing arms including C6.5, C6ML3-9, C6 MH3-B1, C6-B1D2, F5, HER3.A5,HER3.F4, HER3.H1, HER3.H3, HER3.E12, HER3.B12, EGFR.E12, EGFR.C10,EGFR.B11, EGFR.E8, HER4.B4, HER4.G4, HER4.F4, HER4.A8, HER4.B6, HER4.D4,HER4.D7, HER4.D11, HER4.D12, HER4.E3, HER4.E7, HER4.F8 and HER4.C7. In aparticularly preferred embodiment, the arms are linked together with alinker molecule having the amino acid sequence of SEQ ID NO: 11. Vectorsand transformants comprising the nucleic acid sequences encoding thescFv arms and the linker molecule are also provided.

An exemplary bs-scFv antibody that has binding affinity for two membersof the EGFR protein family is ALM which has one arm that has bindingspecificity for HER3 and a second arm that has binding specificity forHER2/neu. An exemplary bs-scFv antibody that has binding affinity fortwo epitopes on a single member of the EGFR protein family is ALF, whichhas one arm with binding specificity for an epitope on HER3 and a secondarm with binding specificity for a different epitope on HER3.

In another embodiment of the invention, the bs-scFv antibodies havebinding affinity for members of the EGFR protein family that areoverexpressed by tumor cells.

In yet another embodiment of the invention, compositions and methods fortreating cancer are provided wherein a patient is administered atherapeutically effective amount of a bs-scFv antibody molecule of theinvention in a pharmaceutically acceptable carrier, either alone or incombination with other cytotoxic agents, such as, chemotherapeuticagents, external beam radiation, targeted radioisotopes and signaltransduction inhibitors.

DEFINITIONS

The terms “polypeptide”, “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical analogue of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers. The term also includes variants on the traditional peptidelinkage joining the amino acids making up the polypeptide. Preferred“peptides”, “polypeptides”, and “proteins” are chains of amino acidswhose a carbons are linked through peptide bonds. The terminal aminoacid at one end of the chain (amino terminal) therefore has a free aminogroup, while the terminal amino acid at the other end of the chain(carboxy terminal) has a free carboxyl group. As used herein, the term“amino terminus” (abbreviated N-terminus) refers to the free α-aminogroup on an amino acid at the amino terminal of a peptide or to theα-amino group (imino group when participating in a peptide bond) of anamino acid at any other location within the peptide. Similarly, the term“carboxy terminus” refers to the free carboxyl group on the carboxyterminus of a peptide or the carboxyl group of an amino acid at anyother location within the peptide. Peptides also include essentially anypolyamino acid including, but not limited to peptide mimetics such asamino acids joined by an ether as opposed to an amide bond.

As used herein, an “antibody” refers to a protein consisting of one ormore polypeptides substantially encoded by immunoglobulin genes orfragments of immunoglobulin genes. The recognized immunoglobulin genesinclude the kappa, lambda, alpha, gamma, delta, epsilon and mu constantregion genes, as well as myriad immunoglobulin variable region genes.Light chains are classified as either kappa or lambda. Heavy chains areclassified as gamma, mu, alpha, delta, or epsilon, which in turn definethe immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.

A typical immunoglobulin (antibody) structural unit is known to comprisea tetramer. Each tetramer is composed of two identical pairs ofpolypeptide chains, each pair having one “light” (about 25 kD) and one“heavy” chain (about 50-70 kD). The N-terminus of each chain defines avariable region of about 100 to 110 or more amino acids primarilyresponsible for antigen recognition. The terms variable light chain(V_(L)) and variable heavy chain (V_(H)) refer to these light and heavychains respectively.

Antibodies exist as intact immunoglobulins or as a number of wellcharacterized fragments produced by digestion with various peptidases.Thus, for example, pepsin digests an antibody below the disulfidelinkages in the hinge region to produce F(ab)′₂, a dimer of Fab whichitself is a light chain joined to V_(H)-C_(H)1 by a disulfide bond. TheF(ab)′₂ may be reduced under mild conditions to break the disulfidelinkage in the hinge region thereby converting the (Fab′)₂ dimer into aFab′ monomer. The Fab′ monomer is essentially a Fab with part of thehinge region (see, Fundamental Immunology, W. E. Paul, ed., Raven Press,N.Y. (1993), for a more detailed description of other antibodyfragments). While various antibody fragments are defined in terms of thedigestion of an intact antibody, one of skill will appreciate that suchFab′ fragments may be synthesized de novo either chemically or byutilizing recombinant DNA methodology. Thus, the term antibody, as usedherein also includes whole antibodies, antibody fragments eitherproduced by the modification of whole antibodies or synthesized de novousing recombinant DNA methodologies. Preferred antibodies include singlechain antibodies (antibodies that exist as a single polypeptide chain),more preferably single chain Fv antibodies (scFv) in which a variableheavy and a variable light chain are joined together (directly orthrough a peptide linker) to form a continuous polypeptide. The singlechain Fv antibody is a covalently linked V_(H-)V_(L) heterodimer whichmay be expressed from a nucleic acid including V_(H)- and V_(L)-encodingsequences either joined directly or joined by a peptide-encoding linker.Huston, et al. (1988) Proc. Nat. Acad. Sci. USA, 85: 5879-5883. Whilethe V_(H) and V_(L) are connected to each as a single polypeptide chain,the V_(H) and V_(L) domains associate non-covalently. The firstfunctional antibody molecules to be expressed on the surface offilamentous phage were single-chain Fv′s (scFv), however, alternativeexpression strategies have also been successful. For example Fabmolecules can be displayed on phage if one of the chains (heavy orlight) is fused to g3 capsid protein and the complementary chainexported to the periplasm as a soluble molecule. The two chains can beencoded on the same or on different replicons; the important point isthat the two antibody chains in each Fab molecule assemblepost-translationally and the dimer is incorporated into the phageparticle via linkage of one of the chains to, e.g., g3p (see, e.g., U.S.Pat. No. 5,733,743). The scFv antibodies and a number of otherstructures converting the naturally aggregated, but chemically separatedlight and heavy polypeptide chains from an antibody V region into amolecule that folds into a three dimensional structure substantiallysimilar to the structure of an antigen-binding site are known to thoseof skill in the art (see e.g., U.S. Pat. Nos. 5,091,513, 5,132,405, and4,956,778). Particularly preferred antibodies should include all thathave been displayed on phage (e.g., scFv, Fv, Fab and disulfide linkedFv (Reiter et al. (1995) Protein Eng. 8: 1323-1331), and also includebispecific, trispecific, quadraspecific, and generally polyspecificantibodies (e.g. bs scFv).

With respect to antibodies of the invention, the term “immunologicallyspecific” “specifically binds” refers to antibodies that bind to one ormore epitopes of a protein of interest (e.g., HER2/neu), but which donot substantially recognize and bind other molecules in a samplecontaining a mixed population of antigenic biological molecules.

The term “bispecific antibody” as used herein refers to an antibodycomprising two antigen-binding sites, a first binding site havingaffinity for a first antigen or epitope and a second binding site havingbinding affinity for a second antigen or epitope distinct from thefirst.

The terms “nucleic acid” or “oligonucleotide” or grammatical equivalentsherein refer to at least two nucleotides covalently linked together. Anucleic acid of the present invention is preferably single-stranded ordouble stranded and will generally contain phosphodiester bonds,although in some cases, as outlined below, nucleic acid analogs areincluded that may have alternate backbones, comprising, for example,phosphoramide (Beaucage et al. (1993) Tetrahedron 49(10):1925) andreferences therein; Letsinger (1970) J. Org. Chem. 35:3800; Sprinzl etal. (1977) Eur. J. Biochem. 81: 579; Letsinger et al. (1986) Nucl. AcidsRes. 14: 3487; Sawai et al. (1984) Chem. Lett. 805, Letsinger et al.(1988) J. Am. Chem. Soc. 110: 4470; and Pauwels et al. (1986) ChemicaScripta 26: 141 9), phosphorothioate (Mag et al. (1991) Nucleic AcidsRes. 19:1437; and U.S. Pat. No. 5,644,048), phosphorodithioate (Briu etal. (1989) J. Am. Chem. Soc. 111:2321, O-methylphosphoroamidite linkages(see Eckstein, Oligonucleotides and Analogues: A Practical Approach,Oxford University Press), and peptide nucleic acid backbones andlinkages (see Egholm (1992) J. Am. Chem. Soc. 114:1895; Meier et al.(1992) Chem. Int. Ed. Engl. 31: 1008; Nielsen (1993) Nature, 365: 566;Carlsson et al. (1996) Nature 380: 207). Other analog nucleic acidsinclude those with positive backbones (Denpcy et al. (1995) Proc. Natl.Acad. Sci. USA 92: 6097; non-ionic backbones (U.S. Pat. Nos. 5,386,023,5,637,684, 5,602,240, 5,216,141 and 4,469,863; Angew. (1991) Chem. Intl.Ed. English 30: 423; Letsinger et al. (1988) J. Am. Chem. Soc. 110:4470;Letsinger et al. (1994) Nucleoside & Nucleotide 13:1597; Chapters 2 and3, ASC Symposium Series 580, “Carbohydrate Modifications in AntisenseResearch”, Ed. Y. S. Sanghui and P. Dan Cook; Mesmaeker et al. (1994),Bioorganic & Medicinal Chem. Lett. 4: 395; Jeffs et al. (1994) J.Biomolecular NMR 34:17; Tetrahedron Lett. 37:743 (1996)) and non-ribosebackbones, including those described in U.S. Pat. Nos. 5,235,033 and5,034,506, and Chapters 6 and 7, ASC Symposium Series 580, CarbohydrateModifications in Antisense Research, Ed. Y. S. Sanghui and P. Dan Cook.Nucleic acids containing one or more carbocyclic sugars are alsoincluded within the definition of nucleic acids (see Jenkins et al.(1995), Chem. Soc. Rev. pp 169-176). Several nucleic acid analogs aredescribed in Rawls, C & E News Jun. 2, 1997 page 35. These modificationsof the ribose-phosphate backbone may be done to facilitate the additionof additional moieties such as labels, or to increase the stability andhalf-life of such molecules in physiological environments.

The terms “hybridizing specifically to” and “specific hybridization” and“selectively hybridize to,” as used herein refer to the binding,duplexing, or hybridizing of a nucleic acid molecule preferentially to aparticular nucleotide sequence under stringent conditions. The term“stringent conditions” refers to conditions under which a probe willhybridize preferentially to its target subsequence, and to a lesserextent to, or not at all to, other sequences. Stringent hybridizationand stringent hybridization wash conditions in the context of nucleicacid hybridization are sequence dependent, and are different underdifferent environmental parameters. An extensive guide to thehybridization of nucleic acids is found in, e.g., Tijssen (1993)Laboratory Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Acid Probes part I, chapt 2, Overviewof principles of hybridization and the strategy of nucleic acid probeassays, Elsevier, N.Y. (Tijssen). Generally, highly stringenthybridization and wash conditions are selected to be about 5° C. lowerthan the thermal melting point (T_(m)) for the specific sequence at adefined ionic strength and pH. The T_(m) is the temperature (underdefined ionic strength and pH) at which 50% of the target sequencehybridizes to a perfectly matched probe. Very stringent conditions areselected to be equal to the T_(m) for a particular probe. An example ofstringent hybridization conditions for hybridization of complementarynucleic acids which have more than 100 complementary residues on anarray or on a filter in a Southern or northern blot is 42° C. usingstandard hybridization solutions (see, e.g., Sambrook (1989) MolecularCloning: A Laboratory Manual (2nd ed.) Vol. 1-3, Cold Spring HarborLaboratory, Cold Spring Harbor Press, NY, and detailed discussion,below), with the hybridization being carried out overnight. An exampleof highly stringent wash conditions is 0.15 M NaCl at 72° C. for about15 minutes. An example of stringent wash conditions is a 0.2×SSC wash at65° C. for 15 minutes (see, e.g., Sambrook supra.) for a description ofSSC buffer). Often, a high stringency wash is preceded by a lowstringency wash to remove background probe signal. An example mediumstringency wash for a duplex of, e.g., more than 100 nucleotides, is1×SSC at 45° C. for 15 minutes. An example of a low stringency wash fora duplex of, e.g., more than 100 nucleotides, is 4× to 6×SSC at 40° C.for 15 minutes.

When applied to RNA, the term “isolated nucleic acid” refers primarilyto an RNA molecule encoded by an isolated DNA molecule as defined above.Alternatively, the term may refer to an RNA molecule that has beensufficiently separated from other nucleic acids with which it would beassociated in its natural state (i.e., in cells or tissues). An“isolated nucleic acid” (either DNA or RNA) may further represent amolecule produced directly by biological or synthetic means andseparated from other components present during its production.

A “replicon” is any genetic element, for example, a plasmid, cosmid,bacmid, plastid, phage or virus, that is capable of replication largelyunder its own control. A replicon may be either RNA or DNA and may besingle or double stranded.

A “vector” is a replicon, such as a plasmid, cosmid, bacmid, phage orvirus, to which another genetic sequence or element (either DNA or RNA)may be attached so as to bring about the replication of the attachedsequence or element.

An “expression operon” refers to a nucleic acid segment that may possesstranscriptional and translational control sequences, such as promoters,enhancers, translational start signals (e.g., ATG or AUG codons),polyadenylation signals, terminators, and the like, and which facilitatethe expression of a polypeptide coding sequence in a host cell ororganism.

The term “primer” as used herein refers to an oligonucleotide, eitherRNA or DNA, either single-stranded or double-stranded, either derivedfrom a biological system, generated by restriction enzyme digestion, orproduced synthetically which, when placed in the proper environment, isable to functionally act as an initiator of template-dependent nucleicacid synthesis. When presented with an appropriate nucleic acidtemplate, suitable nucleoside triphosphate precursors of nucleic acids,a polymerase enzyme, suitable cofactors and conditions such asappropriate temperature and pH, the primer can be extended at its 3′terminus by the addition of nucleotides by the action of a polymerase orsimilar activity to yield a primer extension product. The primer canvary in length depending on the particular conditions and requirement ofthe application. Often primers range from about 15 to about 25 or morenucleotides in length. The primer are typically of sufficientcomplementarity to the desired template to prime the synthesis of thedesired extension product. In other words, the primers are able toanneal with the desired template strand in a manner sufficient toprovide the 3′ hydroxyl moiety of the primer in appropriatejuxtaposition for use in the initiation of synthesis by a polymerase orsimilar enzyme. It is not required that the primer sequence represent anexact complement of the desired template. For example, anon-complementary nucleotide sequence may be attached to the 5′ end ofan otherwise complementary primer. Alternatively, non-complementarybases can be interspersed within the oligonucleotide primer sequence,provided that the primer sequence has sufficient complementarity withthe sequence of the desired template strand to functionally provide atemplate-primer complex for the synthesis of the extension product.

Polymerase chain reaction (PCR) has been described in U.S. Pat. Nos.4,683,195, 4,800,195, and 4,965,188, the entire disclosures of which areincorporated by reference herein.

As used herein, the terms “reporter,” “reporter system”, “reportergene,” or “reporter gene product” shall mean an operative genetic systemin which a nucleic acid comprises a gene that encodes a product thatwhen expressed produces a reporter signal that is a readily measurable,e.g., by biological assay, immunoassay, radio immunoassay, or bycolorimetric, fluorogenic, chemiluminescent or other methods. Thenucleic acid can be either RNA or DNA, linear or circular, single ordouble stranded, antisense or sense polarity, and is operatively linkedto the necessary control elements for the expression of the reportergene product. The control elements will vary according to the nature ofthe reporter system and whether the reporter gene is in the form of DNAor RNA, but may include, but not be limited to, such elements aspromoters, enhancers, translational control sequences, poly A additionsignals, transcriptional termination signals and the like.

The terms “transform”, “transfect”, “transduce”, shall refer to anymethod or means by which a nucleic acid is introduced into a cell orhost organism and may be used interchangeably to convey the samemeaning. Such methods include, but are not limited to, transfection,electroporation, microinjection, PEG-fusion and the like. The introducednucleic acid may or may not be integrated (covalently linked) intonucleic acid of the recipient cell or organism. In bacterial, yeast,plant and mammalian cells, for example, the introduced nucleic acid maybe maintained as an episomal element or independent replicon such as aplasmid. Alternatively, the introduced nucleic acid may becomeintegrated into the nucleic acid of the recipient cell or organism andbe stably maintained in that cell or organism and further passed on orinherited to progeny cells or organisms of the recipient cell ororganism. Finally, the introduced nucleic acid may exist in therecipient cell or host organism only transiently.

The term “selectable marker gene” refers to a gene that when expressedconfers a selectable phenotype, such as antibiotic resistance, on atransformed cell or plant.

The term “operably linked” means that the regulatory sequences necessaryfor expression of the coding sequence are placed in the DNA molecule inthe appropriate positions relative to the coding sequence so as toeffect expression of the coding sequence. This same definition issometimes applied to the arrangement of transcription units and othertranscription control elements (e.g. enhancers) in an expression vector.

The terms “identical” or percent “identity,” in the context of two ormore nucleic acids or polypeptide sequences, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same, whencompared and aligned for maximum correspondence, as measured using oneof the following sequence comparison algorithms or by visual inspection.With respect to the peptides of this invention sequence identity isdetermined over the full length of the peptide.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are input into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. The sequencecomparison algorithm then calculates the percent sequence identity forthe test sequence(s) relative to the reference sequence, based on thedesignated program parameters.

Optimal alignment of sequences for comparison can be conducted, e.g., bythe local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482(1981), by the homology alignment algorithm of Needleman & Wunsch, J.Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson& Lipman (1988) Proc. Natl. Acad. Sci. USA 85:2444, by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group, 575Science Dr., Madison, Wis.), or by visual inspection (see generallyAusubel et al., supra).

One example of a useful algorithm is PILEUP. PILEUP creates a multiplesequence alignment from a group of related sequences using progressive,pairwise alignments to show relationship and percent sequence identity.It also plots a tree or dendrogram showing the clustering relationshipsused to create the alignment. PILEUP uses a simplification of theprogressive alignment method of Feng & Doolittle (1987) J. Mol. Evol.35:351-360. The method used is similar to the method described byHiggins & Sharp (1989) CABIOS 5: 151-153. The program can align up to300 sequences, each of a maximum length of 5,000 nucleotides or aminoacids. The multiple alignment procedure begins with the pairwisealignment of the two most similar sequences, producing a cluster of twoaligned sequences. This cluster is then aligned to the next most relatedsequence or cluster of aligned sequences. Two clusters of sequences arealigned by a simple extension of the pairwise alignment of twoindividual sequences. The final alignment is achieved by a series ofprogressive, pairwise alignments. The program is run by designatingspecific sequences and their amino acid or nucleotide coordinates forregions of sequence comparison and by designating the programparameters. For example, a reference sequence can be compared to othertest sequences to determine the percent sequence identity relationshipusing the following parameters: default gap weight (3.00), default gaplength weight (0.10), and weighted end gaps.

Another example of algorithm that is suitable for determining percentsequence identity and sequence similarity is the BLAST algorithm, whichis described in Altschul et al. (1990) J. Mol. Biol. 215: 403-410.Software for performing BLAST analyses is publicly available through theNational Center for Biotechnology Information(http://www.ncbi.nlm.nih.gov/). This algorithm involves firstidentifying high scoring sequence pairs (HSPs) by identifying shortwords of length W in the query sequence, which either match or satisfysome positive-valued threshold score T when aligned with a word of thesame length in a database sequence. T is referred to as the neighborhoodword score threshold (Altschul et al, supra). These initial neighborhoodword hits act as seeds for initiating searches to find longer HSPscontaining them. The word hits are then extended in both directionsalong each sequence for as far as the cumulative alignment score can beincreased. Cumulative scores are calculated using, for nucleotidesequences, the parameters M (reward score for a pair of matchingresidues; always >0) and N (penalty score for mismatching residues;always <0). For amino acid sequences, a scoring matrix is used tocalculate the cumulative score. Extension of the word hits in eachdirection are halted when: the cumulative alignment score falls off bythe quantity X from its maximum achieved value; the cumulative scoregoes to zero or below, due to the accumulation of one or morenegative-scoring residue alignments; or the end of either sequence isreached. The BLAST algorithm parameters W, T, and X determine thesensitivity and speed of the alignment. The BLASTN program (fornucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) of 10, M=5, N=−4, and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a wordlength(W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (seeHenikoff & Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915).

In addition to calculating percent sequence identity, the BLASTalgorithm also performs a statistical analysis of the similarity betweentwo sequences (see, e.g., Karlin & Altschul (1993) Proc. Natl. Acad.Sci. USA, 90: 5873-5787). One measure of similarity provided by theBLAST algorithm is the smallest sum probability (P(N)), which providesan indication of the probability by which a match between two nucleotideor amino acid sequences would occur by chance. For example, a nucleicacid is considered similar to a reference sequence if the smallest sumprobability in a comparison of the test nucleic acid to the referencenucleic acid is less than about 0.1, more preferably less than about0.01, and most preferably less than about 0.001.

The phrase “specifically target/deliver” when used, for example withreference to a chimeric moiety of this invention refers to specificbinding of the moiety to a target (e.g. a cell overexpressing the targetprotein(s)) this results in an increase in local duration and/orconcentration of the moiety at or within the cell as compared to thatwhich would be obtained without “specific” targeting. The specificityneed not be absolute, but simply detectably greater/measurablyavidity/affinity than that observed for a cell expressing the targetprotein(s) at normal (e.g., wildtype) or than that observed for a cellthat does not express the target protein(s).

Amino acid residues are identified in the present application accordingto standard 3-letter or 1-letter abbreviations (e.g. as set forth inWIPO standard ST 25) and/or as set forth in Table 1.

TABLE 1 Amino acid abbreviations. 3 Letter 1 Letter Amino AcidAbbreviation Abbreviation L-Alanine Ala A L-Arginine Arg R L-AsparagineAsn N L-AsparticAcid Asp D L-Cysteine Cys C L-Glutamine Gln QL-GlutamicAcid Glu E Glycine Gly G L-Histidine His H L-Isoleucine Ile IL-Leucine Leu L L-Methionine Met M L-Phenylalanine Phe F L-Proline Pro PL-Serine Ser S L-Threonine Thr T L-Tryptophan Trp W L-Tyrosine Tyr YL-Valine Val V L-Lysine Lys K

Enantiomeric amino acids described herein are preferred to be in the “L”isomeric form. However, residues in the “D” isomeric form can besubstituted for any L-amino acid residue, provided the desiredproperties of the polypeptide are retained. All amino-acid residuesequences represented herein conform to the conventional left-to-rightamino-terminus to carboxy-terminus orientation.

The term “isolated protein” or “isolated and purified protein” issometimes used herein. This term refers primarily to a protein producedby expression of an isolated nucleic acid molecule of the invention.Alternatively, this term may refer to a protein that has beensufficiently separated from other proteins with which it would naturallybe associated, so as to exist in “substantially pure” form. “Isolated”is not meant to exclude artificial or synthetic mixtures with othercompounds or materials, or the presence of impurities that do notinterfere with the fundamental activity, and that may be present, forexample, due to incomplete purification, addition of stabilizers, orcompounding into, for example, immunogenic preparations orpharmaceutically acceptable preparations.

The term “substantially pure” refers to a preparation comprising atleast 50-60% by weight of a given material (e.g., nucleic acid,oligonucleotide, protein, etc.). More preferably, the preparationcomprises at least 75% by weight, and most preferably 90-95% by weightof the given compound. Purity is measured by methods appropriate for thegiven compound (e.g. chromatographic methods, agarose or polyacrylamidegel electrophoresis, HPLC analysis, and the like).

The term “functional” as used herein implies that the nucleic or aminoacid sequence is functional for the recited assay or purpose.

The phrase “consisting essentially of” when referring to a particularnucleotide or amino acid means a sequence having the properties of agiven SEQ ID NO. For example, when used in reference to an amino acidsequence, the phrase includes the sequence per se and molecularmodifications that would not affect the basic and novel characteristicsof the sequence.

The term “tag,” “tag sequence” or “protein tag” refers to a chemicalmoiety, either a nucleotide, oligonucleotide, polynucleotide or an aminoacid, peptide or protein or other chemical, that when added to anothersequence, provides additional utility or confers useful properties,particularly in the detection or isolation, of that sequence. Thus, forexample, a homopolymer nucleic acid sequence or a nucleic acid sequencecomplementary to a capture oligonucleotide may be added to a primer orprobe sequence to facilitate the subsequent isolation of an extensionproduct or hybridized product. In the case of protein tags, histidineresidues (e.g., 4 to 8 consecutive histidine residues) may be added toeither the amino- or carboxy-terminus of a protein to facilitate proteinisolation by chelating metal chromatography. Alternatively, amino acidsequences, peptides, proteins or fusion partners representing epitopesor binding determinants reactive with specific antibody molecules orother molecules (e.g., flag epitope, c-myc epitope, transmembraneepitope of the influenza A virus hemaglutinin protein, protein A,cellulose binding domain, calmodulin binding protein, maltose bindingprotein, chitin binding domain, glutathione S-transferase, and the like)may be added to proteins to facilitate protein isolation by proceduressuch as affinity or immunoaffinity chromatography. Chemical tag moietiesinclude such molecules as biotin, which may be added to either nucleicacids or proteins and facilitates isolation or detection by interactionwith avidin reagents, and the like. Numerous other tag moieties areknown to, and can be envisioned by the trained artisan, and arecontemplated to be within the scope of this definition.

A “clone” or “clonal cell population” is a population of cells derivedfrom a single cell or common ancestor, e.g., by mitosis.

A “cell line” is a clone of a primary cell or cell population that iscapable of stable growth in vitro for many generations.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a schematic diagram of the pUC/ALM vector.

FIG. 2 shows a graph illustrating the binding of ALM proteins to theHER3 extracellular domain on a BIAcore chip.

FIG. 3 shows a graph illustrating the binding of ALM proteins to theHER2/neu extracellular domain on a BIAcore chip.

FIG. 4 shows a graph illustrating the simultaneous binding of ALMproteins to HER3 and HER2/neu on a BIAcore chip.

FIGS. 5A and 5B show graphs of flow cytometry results displaying areduction in cell surface HER2/neu and HER3 following in vitroincubation of ALM with human BT-474 breast cancer cells expressing bothHER2/neu and HER3.

FIG. 6 Shows a graph of the results of an MTT assay demonstrating thatALM diminishes proliferation of BT-474 breast cancer cells expressingboth HER2/neu and HER3.

FIG. 7 shows a graph illustrating the results of a 17 day clonogenicityassay demonstrating that incubation of BT-474 cells with ALM at aconcentration that is equimolar with cell surface HER2/neu expressionleads to a 50% reduction in colony formation (cell survival).

FIG. 8 shows a western blot analysis exhibiting alterations inphosphorylation of AKT over 48 hours following in vitro incubation ofdifferent concentrations of ALM with human BT-474 breast cancer cellsexpressing both HER2/neu and HER3.

FIG. 9 shows a graph mapping the biodistribution of 1-labeled ALM over48 hours in immunodeficient mice.

FIG. 10 illustrates a chelate ²¹¹At-SAPS used to label a bispecificantibody according to this invention.

FIGS. 11A through 11C show the effects of ²¹¹At-conjugated ALM at lowdosage of 10 μg (FIG. 11C) and at high a dose of 80 μg (FIG. 11B) ascompared to untreated controls (FIG. 11A).

FIG. 12 illustrates specific tumor labeling in a mouse using a¹²⁴I-labeled bispecific antibody (ALM). PET(upper right) and CT (upperleft) images of scid mice with SK-OV-3 ovarian carcinoma xenograftexpressing HER2/neu and HER3 antigens and imaged 48 hours post-injectionon a G.E. Discovery LS at FCCC. The CT slide thickness is 0.63 mm. Imagefusion (lower right) performed with MIM software.

FIG. 13 shows the sequences of ScFv light and heavy chains as determinedby the Adams Lab and used in the construction of certain bs-scFvmolecules. Sequences are given for C6.5 heavy chain (SEQ ID NO:38), G98Aheavy chain (SEQ ID NO:39), ML3-9 heavy chain (SEQ ID NO:40), H3B1 heavychain (SEQ ID NO:41), B1D2 heavy chain (SEQ ID NO:42), C6.5 light chain(SEQ ID NO:43), G98A light chain (SEQ ID NO:44), ML3-9 light chain (SEQID NO:45), H3B1 light chain (SEQ ID NO:46), and B1D2 light chain (SEQ IDNO:47).

FIG. 14 shows deduced protein sequences of heavy and light chainvariable regions of all IgG produced in IDEC vector provided by theMarks lab. In certain embodiments the sequence of scFvs can differ fromthis. Sequences are given for C6.5 heavy chain (SEQ ID NO:48), G98Aheavy chain (SEQ ID NO:49), ML3-9 heavy chain (SEQ ID NO:50), H3B1 heavychain (SEQ ID NO:51), B1D2 heavy chain (SEQ ID NO:52), C6.5 light chain(SEQ ID NO:53), G98A light chain (SEQ ID NO:54), ML3-9 light chain (SEQID NO:55), H3B1 light chain (SEQ ID NO:56), and B1D2 light chain (SEQ IDNO:57).

DETAILED DESCRIPTION

Tumors often overexpress growth factor receptors that bind variousligands ligand and facilitate unrestricted tumor growth. One example ofsuch growth factor receptors is the Epidermal Growth Factor Receptor(EGFR) protein family.

Signal transduction through members of the Epidermal Growth FactorReceptor (EGFR) protein family is dependent upon the formation ofhomodimers or heterodimers triggered by the binding of ligand. Thisreceptor family is comprised of four membrane-bound proteins: EGFR,HER2/neu, HER3 and HER4. Overexpression of these proteins has beencorrelated with a poor prognosis in a number of types of cancer,including, but not limited to, breast, colon, ovarian, endometrial,gastric, pancreatic, prostate and salivary gland cancers. While a numberof groups have developed strategies to target individual members of theEGFR protein family (e.g., HER2/neu or EGFR) to inhibit tumor growth,none of the treatments has been proven to ultimately cure these forms ofcancer.

In accordance with the present invention, novel antibody constructs havebeen developed that are capable of simultaneously targeting multiplemembers multiple members (or multiple sites on a given member) of theEGFR protein family. The antibody constructs typically comprise a firstantibody and a second antibody joined to each other where the firstantibody and the second antibody bind specifically to different epitopeson the same or different members of the EGFR protein family. In certainembodiments, the bispecific antibody constructs are bispecific singlechain molecules (e.g., bispecific single chain Fv (bs-scFv)), but theconstructs need not be so limited. Thus, for example, chemicallyconjugated whole antibodies, or antibody fragments are also contemplatedwithin the scope of this invention. In general, where bi-specificantibodies are described herein, it will be appreciated thattrispecific, or more generally polyspecific antibodies are alsocontemplated.

The bispecific antibodies of this invention bind to selected members ofthe EGFR protein family (e.g., EGFR, HER2/neu, HER3, HER4) to preventligand induced signaling and/or to trigger cytostatic and/or cytotoxiceffects. The bispecific antibodies can also be used to specificallylabel cancer cells, solid tumors, and the like, and, more generally, tospecifically target/deliver any conjugated or otherwise coupled effector(e.g. radioisotope, label, cytotoxin, drug, liposome, antibody, nucleicacid, dendrimer, etc.) to cancer cells including but not limited toisolated cancer cells, metastatic cells, solid tumor cells, and thelike.

In certain preferred embodiments, the bispecific antibodies of thisinvention are bispecific single chain Fv antibodies (bs-scFv). Singlechain Fv antibody fragments are engineered antibody derivatives thatinclude both a heavy and a light chain variable region joined by apeptide linker molecule and are potentially more effective thanunmodified IgG antibodies because their reduced size permits them topenetrate tissues and solid tumors more readily than IgG antibodies.

In one embodiment the bispecific antibodies of this invention (e.g. thebs-scFv antibody molecules) comprise two domains that provide twodistinct binding specificities. A first domain has binding specificityfor an epitope on one member of the EGFR protein family and the seconddomain has binding specificity for an epitope on a second member of theEGFR protein family. An exemplary bs-scFv molecule of the invention is“ALM”; a bispecific antibody that was created with one arm (domain) thatexhibits binding specificity to an epitope on HER2/neu and a second arm(domain) that exhibits binding specificity to an epitope on HER3.

Alternatively, the bispecific antibodies of the invention can begenerated such that one domain has binding specificity for one epitopeon a member of the EGFR protein family and a second domain has bindingspecificity for a second distinct epitope on the same member of the EGFRprotein family. An exemplary bs-scFv of this type is “ALF” which iscomposed of two distinct scFV molecules, both with a specificity forHER3.

I. Antibodies Forming the Bispecific or Polyspecific Antibodies of thisInvention.

As indicated above, the bispecific or polyspecific antibodies of thisinvention typically comprise two or more binding domains at least two ofwhich are specific to different epitopes of the EGFR protein family.Preferred antibodies of this invention comprise domains specific toepitopes of EGFR, HER2/neu, HER3 and HER4.

Using phage display approaches, a number of single chain antibodies havebeen raised that are specific to various epitopes on these members ofthe EGFR protein family. These single chain Fv antibodies can be used asdomains/arms to construct a bispecific or polyspecific antibodyaccording to this invention. A number of these antibodies are provided,below, in Table 2 and in FIGS. 13 and 14. Each arm (antibody) can bepaired with a different arm to form either a bs-scFv antibody withbinding specificity for two distinct epitopes on different members ofthe EGFR protein family or a bs-scFv antibody with binding specificityfor two distinct epitopes on the same member of the EGFR protein family.

TABLE 2 Single-chain Fv antibodies directed against epitopes of the EGFRprotein family. Anti-HER2/neu*: Anti-HER3**: C6.5*** HER3.A5 C6ML3-9(ML3.9 or C6ML3.9) HER3.F4 (SEQ ID NO: 2 protein, C6MH3-B1 (B1 orC6MH3.B1) SEQ ID NO: 28 DNA) C6-B1D2 (B1D2 or C6MH3-B1D2) HER3.H1 (SEQID NO: 3 protein, F5 (SEQ ID NO: 1 protein, SEQ ID SEQ ID NO: 29 DNA)NO: 27 DNA)** HER3.H3 (SEQ ID NO: 4 protein, HER3.B12 (SEQ ID NO: 6protein, SEQ ID NO: 30 DNA)) SEQ ID NO: 32 DNA) HER3.E12 (SEQ ID NO: 5protein, SEQ ID NO: 31 DNA)) Anti-EGFR**: Anti-HER4: EGFR.E12 (SEQ IDNO: 7 protein, HER4.B4 SEQ ID NO: 33 DNA) HER4.G4 EGFR.C10 (SEQ ID NO: 8protein, HER4.F4 SEQ ID NO: 34 DNA) HER4.A8 EGFR.B11 (SEQ ID NO: 9protein, HER4.B6 (SEQ ID NO: 19 SEQ ID NO: 35 DNA) protein, SEQ ID NO:37 DNA) EGFR.E8 (SEQ ID NO: 10 protein, HER4.D4 SEQ ID NO: 36 DNA)HER4.D7 HER4.D11 HER4.D12 HER4.E3 (SEQ ID NO: 21 protein, SEQ ID NO: 38DNA) HER4.E7 HER4.F8 HER4.C7 *Sequences are disclosed in Schier et al.(1996). J. Mol. Biol., 255(1): 28-43. See also Schier et al. (1995)Immunotechnology, 1: 73-81. **Sequences are provided in Appendix Ahereinbelow; ***Sequences are also shown in FIGS. 13 and 14.

The bispecific or polyspecific antibodies of this invention, howeverneed not be limited to the use of the particular antibodies enumeratedin Table 2 and/or FIG. 13 or 14. In effect, each of the antibodieslisted in Table 2 and/or FIG. 13 or 14 identifies an epitope of a memberof the EGFR protein family and these antibodies can readily be used toidentify other antibodies that bind to the same epitopes. Thus, incertain embodiments, the bispecific or polyspecific antibodies of thisinvention comprise one or more domains that specifically bind an epitopespecifically bound by an antibody of Table 2 and/or FIG. 13 or 14 (e.g.,an antibody selected from the group consisting of C6.5, C6ML3-9, C6MH3-B1, C6-B1D2, F5, HER3.A5, HER3.F4, HER3.H1, HER3.H3, HER3.E12,HER3.B12, EGFR.E12, EGFR.C10, EGFR.B11, EGFR.E8, HER4.B4, HER4.G4,HER4.F4, HER4.A8, HER4.B6, HER4.D4, HER4.D7, HER4.D11, HER4.D12,HER4.E3, HER4.E7, HER4.F8 and HER4.C7).

Such antibodies are readily identified by screening whole antibodies,antibody fragments, or single chain antibodies for their ability tocompete with the antibodies listed in Table 2 for their ability to bindto a protein comprising the target epitope. In other words, candidateantibodies can be screened for cross-reactivity with the antibodieslisted in Table 2 and/or FIG. 13 or 14 against the target protein in theEGFR protein family.

In a preferred embodiment, the antibodies of this invention specificallybind to one or more epitopes recognized by antibodies listed in Table 2and/or FIG. 13 or 14. In other words, particularly preferred antibodiesare cross-reactive with one of more of these antibodies. Means ofassaying for cross-reactivity are well known to those of skill in theart (see, e.g., Dowbenko et al. (1988) J. Virol. 62: 4703-4711).

For example, in certain embodiments, cross-reactivity can be ascertainedby providing an isolated EGFR family member (e.g., EGFR, HER2/neu, HER3and HER4 or a fragment thereof) attached to a solid support and assayingthe ability of a test antibody to compete with one or more of theantibodies listed in Table 2 for binding to the target protein. Thus,immunoassays in a competitive binding format are can be used forcrossreactivity determinations. For example, in one embodiment, the EGFRfamily member polypeptide is immobilized to a solid support. Antibodiesto be tested (e.g. generated by selection from a phage-display library,or generated in a whole antibody library) are added to the assay competewith one or more of the antibodies listed in Table 2 and/or FIG. 13 or14 for binding to the immobilized polypeptide. The ability of testantibodies to compete with the binding of the antibodies of Table 2and/or FIG. 13 or 14 to the immobilized protein are compared. Thepercent crossreactivity above proteins can then calculated, usingstandard calculations. If the test antibody competes with one or more ofthe Table 2 and/or FIG. 13 or 14 antibodies and has a binding affinitycomparable to or greater than about 1×10⁻⁸ M, more preferably greaterthan 1×10⁻⁹, or 1×10⁻¹⁰, or more generally with an affinity equal to orgreater than the corresponding (competing) antibody, e.g., of Table 2then the antibody is well suited for use in the present invention.

In a particularly preferred embodiment, cross-reactivity is performed byusing surface plasmon resonance in a BIAcore. In a BIAcore flow cell,the EGFR protein is coupled to a sensor chip. With a typical flow rateof 5 (1/min, a titration of 100 nM to 1 (M antibody is injected over theflow cell surface for about 5 minutes to determine an antibodyconcentration that results in near saturation of the surface. Epitopemapping or cross-reactivity is then evaluated using pairs of antibodiesat concentrations resulting in near saturation and at least 100 RU ofantibody bound. The amount of antibody bound is determined for eachmember of a pair, and then the two antibodies are mixed together to givea final concentration equal to the concentration used for measurementsof the individual antibodies. Antibodies recognizing different epitopesshow an essentially additive increase in the RU bound when injectedtogether, while antibodies recognizing identical epitopes show only aminimal increase in RU. In a particularly preferred embodiment,antibodies are said to be cross-reactive if, when “injected” togetherthey show an essentially additive increase (preferably an increase by atleast a factor of about 1.4, more preferably an increase by at least afactor of about 1.6, and most preferably an increase by at least afactor of about 1.8 or 2.

Cross-reactivity at the epitopes recognized by the antibodies listed inTable 2 and/or FIG. 13 or 14 can ascertained by a number of otherstandard techniques (see, e.g., Geysen et al (1987) J. Immunol. Meth.102: 259-274).

In addition, number of the antibodies identified in Table 2 have beensequenced (see, e.g., FIGS. 13 and 14). The amino acid sequencescomprising the complementarity determining regions (CDRs) are thereforeknown. Using this sequence information, the same or similarcomplementarity determining regions can be engineered into otherantibodies to produce chimeric full size antibodies and/or antibodyfragments, e.g. to ensure species compatibility, to increase serumhalf-life, and the like. A large number of methods of generatingchimeric antibodies are well known to those of skill in the art (see,e.g., U.S. Pat. Nos. 5,502,167, 5,500,362, 5,491,088, 5,482,856,5,472,693, 5,354,847, 5,292,867, 5,231,026, 5,204,244, 5,202,238,5,169,939, 5,081,235, 5,075,431, and 4,975,369).

In short, using routine methods, the antibodies listed in Table 2 canreadily be used to generate or identify other antibodies (full length,antibody fragments, single-chain, and the like) that bind to the sameepitope. Similarly, the antibodies listed in Table 2 can readily beutilized to generate other antibodies that have the same or similarcomplementarity determining regions (CDRs).

II. Preparation of Bi-Specific Antibody Molecules:

The antibodies directed to epitopes found on members of the EGFR proteinfamily (e.g. the antibodies listed in Table 2) can be used to preparebispecific or polyspecific antibodies of this invention. The two (ormore) antibodies can be prepared using a variety of methods. Forexample, the antibodies can be prepared separately (e.g. using chemicalprotein synthesis, recombinant expression methods, hybridoma technology,etc.) and then chemically attached to each other, either directly orthrough a linker. Where both antibodies are single chain antibodieseither directly joined at the termini or through a peptide linker, thebispecific or polyspecific molecule can be chemically synthesized, ormore preferably is recombinantly expressed.

Means of chemically conjugating molecules are well known to those ofskill in the art. The procedures for chemically coupling two antibodiesare straightforward. Polypeptides typically contain variety offunctional groups; e.g., carboxylic acid (COOH) or free amine (—NH₂)groups, that are available for reaction with a suitable functionalgroups on the corresponding antibody or on a linker.

Alternatively, the antibodies can be derivatized to expose or attachadditional reactive functional groups. The derivatization can involveattachment of any of a number of linker molecules such as thoseavailable from Pierce Chemical Company, Rockford Ill. A variety ofsuitable linkers are known to those of skill in the art (see, e.g.,European Patent Application No. 188,256; U.S. Pat. Nos. 4,671,958,4,659,839, 4,414,148, 4,699,784; 4,680,338; 4,569,789; and 4,589,071;and Borlinghaus et al. (1987) Cancer Res. 47: 4071-4075) and suitablelinkers are also described below with respect to the coupling ofeffectors to bispecific antibodies.

In certain preferred embodiments of the invention, the bs-scFv antibodymolecules are produced by expression of recombinant antibody fragmentsproduced in host cells. The genes for several of the scFv molecules thattarget various epitopes on members of the EGFR protein family have beencloned (see, e.g., Appendix A and Schier et al. (1996) J. Mol. Biol.,(1): 28-43) and pairs (or other combinations) of these scFv genes can beoperably linked directly or via a linker molecule. The resulting nucleicacid molecules encoding the bs-scFv antibody fragments are inserted intoexpression vectors and introduced into host cells. The resulting bs-scFvantibody molecules are then isolated and purified from the expressionsystem.

In certain preferred embodiments of the invention, the scFv antibodymolecules are paired together with a novel linker molecule designed toprotect against proteolytic degradation of the bs-scFv antibodymolecules. This linker typically lacks a proteolytic cleavage site andis typically characterized by containing primarily neutral (non-charged)amino acids. One such linker sequence has the sequence: Asn Ser Gly AlaGly Thr Ser Gly Ser Gly Ala Ser Gly Glu Gly Ser Gly Ser Lys Leu (SEQ IDNO:37).

The scFv provided in Table 2 are incorporated into new bs-scFv basedupon the following factors: (1) descending affinity for a given target,(2) the lack of cross-reactive epitopes (as determined by bindinginhibition and sandwich assays on a BIAcore), (3) combinations thattarget EGFR family member pairs that have not yet been paired, and (4)inclusion of scFv arms that have led to growth inhibition and alteredsignal transduction when employed in other bs-scFv combinations.

The purity of the bs-scFv antibody molecules of the invention may beassessed using standard methods known to those of skill in the art,including, but not limited to, ELISA, immunohistochemistry, ion-exchangechromatography, affinity chromatography, immobilized metal affinitychromatography (IMAC), size exclusion chromatography, polyacrylamide gelelectrophoresis (PAGE), western blotting, surface plasmon resonance andmass spectroscopy.

Using the antibodies, nucleic acid sequences, and other teachingprovided herein, bispecific or polyspecific antibodies of this inventioncan be recombinantly expressed using routine methods such as those setforth in Sambrook et al. (1989) Molecular Cloning, Cold Spring HarborLaboratory, or Ausubel et al. (eds) (1997) Current Protocols inMolecular Biology, John Wiley & Sons N.Y. In addition illustrativemethods of producing recombinant bispecific single chain antibodies ofthis invention are set forth in the Examples. To the extent thatspecific materials are mentioned, it is merely for purposes ofillustration and is not intended to limit the invention.

III. Chimeric Moieties Comprising Bispecific and/or PolyspecificAntibodies.

In many embodiments, the bispecific and/or polyspecific anti-EGFR familymember antibodies of this invention are capable of inhibiting cancercell growth and/or proliferation without the use of any additional“effector”, in certain embodiments, the bispecific and/or polyspecificantibodies are additionally coupled to an effector thereby formingchimeric moieties that preferentially target/deliver the effector to acell overexpressing the EGFR family member or members.

Since EGFR proteins are often found in upregulated in cancer cells,these proteins can be can be exploited as target(s) for the efficientand specific delivery of an effector (e.g. an effector molecule such asa cytotoxin, a radiolabel, etc.) to various cancer cells (e.g. isolatedcells, metastatic cells, solid tumor cells, etc.), in particular toepithelial cancer cells (e.g. breast cancer cells). The target EGFRprotein(s) need not exist solely on cancer cells to provide an effectivetarget. Differential expression of EGFR on cancer cells, as compared tohealthy cells, is sufficient to provide significant and useful targetingadvantage, i.e. resulting in preferential delivery of the effectormoiety to and/or into the target (e.g. cancer) cell.

In certain preferred embodiments, the bispecific or polyspecificantibodies of this invention are utilized in a “pretargeting” strategy(resulting in formation of a chimeric moiety at the target site afteradministration of the effector moiety) or in a “targeting” strategywhere the bispecific and/or polyspecific antibody is coupled to aneffector molecule prior to use to provide a chimeric moiety.

A chimeric molecule or chimeric composition or chimeric moiety refers toa molecule or composition wherein two or more molecules or compositionsthat exist separately in their native state are joined together to forma single molecule moiety or composition having the desired functionalityof its constituent members. Typically, one of the constituent moleculesof a chimeric moiety is a “targeting molecule”. i.e., in the presentcase a bispecific or polyspecific antibody that specifically binds oneor more members of the EGFR family.

Another constituent of the chimeric molecule is an “effector”. Theeffector molecule refers to a molecule or group of molecules that is tobe specifically transported to the target cell (e.g., a celloverexpressing an EGFR family member). The effector molecule typicallyhas a characteristic activity that is to be delivered to the targetcell. Effector molecules include, but are not limited to cytotoxins,labels, radionuclides, ligands, antibodies, drugs, liposomes, and thelike.

In certain embodiments, the effector is a detectable label, withpreferred detectable labels including radionuclides. Among theradionuclides and labels useful in the radionuclide-chelator-(e.g.biotin) conjugates of the present invention, gamma-emitters,positron-emitters, x-ray emitters and fluorescence-emitters are suitablefor localization, diagnosis and/or staging, and/or therapy, while betaand alpha-emitters and electron and neutron-capturing agents, such asboron and uranium, also can be used for therapy.

The detectable labels can be used in conjunction with an externaldetector and/or an internal detector and provide a means of effectivelylocalizing and/or visualizing, e.g. cancer cells overexpressing one ormore EGFR family members. Such detection/visualization can be useful invarious contexts including, but not limited to pre-operative andintraoperative settings. Thus, in certain embodiment this inventionrelates to a method of intraoperatively detecting and locating tissueshaving EGFR family markers in the body of a mammal. These methodstypically involve administering to the mammal a composition comprising,in a quantity sufficient for detection by a detector (e.g. a gammadetecting probe), a bispecific and/or polyspecific antibody of thisinvention labeled with a detectable label (e.g., antibodies of thisinvention labeled with a radioisotope, e.g. ¹⁶¹Tb, ¹²³I, ¹²⁵I, and thelike), and, after allowing the active substance to be taken up by thetarget tissue, and preferably after blood clearance of the label,subjecting the mammal to a radioimmunodetection technique in therelevant area of the body, e.g. by using a gamma detecting probe.

The label-bound a bispecific and/or polyspecific antibody can be used inthe technique of radioguided surgery, wherein relevant tissues in thebody of a subject can be detected and located intraoperatively by meansof a detector, e.g. a gamma detecting probe. The surgeon can,intraoperatively, use this probe to find the tissues in which uptake ofthe compound labeled with a radioisotope, that is, e.g. a low-energygamma photon emitter, has taken place.

In addition to detectable labels, preferred effectors include cytotoxins(e.g. Pseudomonas exotoxin, ricin, abrin, Diphtheria toxin, and thelike), or cytotoxic drugs or prodrugs, in which case the chimeric moietycan act as a potent cell-killing agent specifically targeting thecytotoxin to cells bearing the EGFR family member(s).

In still other embodiments, the effector can include a liposomeencapsulating a drug (e.g. an anti-cancer drug such as doxirubicin,vinblastine, taxol, etc.), an antigen that stimulates recognition of thebound cell by components of the immune system, an antibody thatspecifically binds immune system components and directs them to thetarget cell(s), and the like.

A) The Bispecific or Polyspecific Anti-EGFR Family Member TargetingMolecule.

In preferred embodiments, of the methods and compositions of thisinvention, the targeting moiety is a bispecific and/or polyspecificantibody that specifically binds to one or more members of the EGFRfamily as described herein. The bispecific and/or polyspecific antibodycan comprise full-length antibodies, antibody fragment(s) (e.g. Fv, Fab,etc.), and/or single chain antibodies (e.g. scFv).

B) Certain Preferred Effectors.

1) Imaging Compositions.

In certain embodiments, the chimeric molecules of this invention can beused to direct detectable labels to a tumor site. This can facilitatetumor detection and/or localization. In certain particularly preferredembodiments, the effector component of the chimeric molecule is a“radiopaque” label, e.g. a label that can be easily visualized usingx-rays. Radiopaque materials are well known to those of skill in theart. The most common radiopaque materials include iodide, bromide orbarium salts. Other radiopaque materials are also known and include, butare not limited to organic bismuth derivatives (see, e.g., U.S. Pat. No.5,939,045), radiopaque polyurethanes (see U.S. Pat. No. 5,346,9810,organobismuth composites (see, e.g., U.S. Pat. No. 5,256,334),radiopaque barium polymer complexes (see, e.g., U.S. Pat. No.4,866,132), and the like.

The a bispecific and/or polyspecific antibodies of this invention) canbe coupled directly to the radiopaque moiety or they can be attached toa “package” (e.g. a chelate, a liposome, a polymer microbead, etc.)carrying or containing the radiopaque material as described below.

In addition to radioopaque labels, other labels are also suitable foruse in this invention. Detectable labels suitable for use as theeffector molecule component of the chimeric molecules of this inventioninclude any composition detectable by spectroscopic, photochemical,biochemical, immunochemical, electrical, optical or chemical means.Useful labels in the present invention include magnetic beads (e.g.Dynabeads™), fluorescent dyes (e.g., fluorescein isothiocyanate, texasred, rhodamine, green fluorescent protein, and the like), radiolabels(e.g., ³H, ¹²⁵I, ³⁵S, ¹⁴C, or ³²P), enzymes (e.g., horse radishperoxidase, alkaline phosphatase and others commonly used in an ELISA),and colorimetric labels such as colloidal gold or colored glass orplastic (e.g. polystyrene, polypropylene, latex, etc.) beads.

Various preferred radiolabels include, but are not limited to ⁹⁹Tc,²⁰³Pb, ⁶⁷Ga, ⁶⁸Ga, ⁷²As, ¹¹¹In, ^(113m)In, ⁹⁷Ru, ⁶²Cu, 641Cu, ⁵²Fe,^(52m)Mn, ⁵¹Cr, ¹⁸⁶Re, ¹⁸⁸Re, ⁷⁷As, ⁹⁰Y, ⁶⁷Cu, ¹⁶⁹Er, ¹²¹Sn, ¹²⁷Te,¹⁴²Pr, ¹⁴³Pr, ¹⁹⁸Au, ¹⁹⁹Au, ¹⁶¹Tb, ¹⁰⁹Pd, ¹⁶⁵Dy, ¹⁴⁹Pm, ¹⁵¹Pm, ¹⁵³Sm,¹⁵⁷Gd, ¹⁵⁹Gd, ¹⁶⁶Ho, ¹⁷²Yb, ¹⁶⁹Yb, ¹⁷⁵Yb, ¹⁷⁷Lu, ¹⁰⁵Rh, and ¹¹¹Ag.

Means of detecting such labels are well known to those of skill in theart. Thus, for example, radiolabels may be detected using photographicfilm, scintillation detectors, and the like. Fluorescent markers may bedetected using a photodetector to detect emitted illumination. Enzymaticlabels are typically detected by providing the enzyme with a substrateand detecting the reaction product produced by the action of the enzymeon the substrate, and colorimetric labels are detected by simplyvisualizing the colored label.

In certain specific embodiments, this invention contemplates the use ofimmunoconjugates (chimeric moieties) for the detection of tumors and/orother cancer cells. Thus, for example, the bispecific antibodies of thisinvention can be conjugated to gamma-emitting radioisotopes (e.g.,Na-22, Cr-51, Co-60, Tc-99, I-125, I-131, Cs-137, GA-67, Mo-99) fordetection with a gamma camera, to positron emitting isotopes (e.g. C-11,N-13, O-15, F-18, and the like) for detection on a Positron EmissionTomography (PET) instrument, and to metal contrast agents (e.g., Gdcontaining reagents, Eu containing reagents, and the like) for magneticresonance imaging (MRI), In addition, the bispecific antibodies of thisinvention can be used in traditional immunohistochemistry (e.g.fluorescent labels, nanocrystal labels, enzymatic and colormetric labelsetc.).

2) Radiosensitizers.

In another embodiment, the effector can be a radiosensitizer thatenhances the cytotoxic effect of ionizing radiation (e.g., such as mightbe produced by ⁶⁰Co or an x-ray source) on a cell. Numerousradiosensitizing agents are known and include, but are not limited tobenzoporphyrin derivative compounds (see, e.g., U.S. Pat. No.5,945,439), 1,2,4-benzotriazine oxides (see, e.g., U.S. Pat. No.5,849,738), compounds containing certain diamines (see, e.g., U.S. Pat.No. 5,700,825), BCNT (see, e.g., U.S. Pat. No. 5,872,107),radiosensitizing nitrobenzoic acid amide derivatives (see, e.g., U.S.Pat. No. 4,474,814), various heterocyclic derivatives (see, e.g., U.S.Pat. No. 5,064,849), platinum complexes (see, e.g., U.S. Pat. No.4,921,963), and the like.

3) Ligands.

The effector molecule may also be a ligand, an epitope tag, or anantibody. Particularly preferred ligand and antibodies are those thatbind to surface markers on immune cells. Chimeric molecules utilizingsuch antibodies as effector molecules act as bifunctional linkersestablishing an association between the immune cells bearing bindingpartner for the ligand or antibody and the tumor cells expressing theEGFR family member(s).

3) Chelates

Many of the pharmaceuticals and/or radiolabels described herein arepreferably provided as a chelate, particularly where a pre-targetingstrategy is utilized. The chelating molecule is typically coupled to amolecule (e.g. biotin, avidin, streptavidin, etc.) that specificallybinds an epitope tag attached to the a bispecific and/or polyspecificantibody.

Chelating groups are well known to those of skill in the art. In certainembodiments, chelating groups are derived from ethylene diaminetetra-acetic acid (EDTA), diethylene triamine penta-acetic acid (DTPA),cyclohexyl 1,2-diamine tetra-acetic acid (CDTA),ethyleneglycol-O,O′-bis(2-aminoethyl)-N,N,N′,N′-tetra-acetic acid(EGTA), N,N-bis(hydroxybenzyl)-ethylenediamine-N,N′-diacetic acid(HBED), triethylene tetramine hexa-acetic acid (TTHA),1,4,7,10-tetraazacyclododecane-N,N′-,N″,N′″-tetra-acetic acid (DOTA),hydroxyethyldiamine triacetic acid (HEDTA),1,4,8,11-tetra-azacyclotetradecane-N,N′,N″,N′″-tetra-acetic acid (TETA),substituted DTPA, substituted EDTA, and the like.

Examples of certain preferred chelators include unsubstituted or,substituted 2-iminothiolanes and 2-iminothiacyclohexanes, in particular2-imino-4-mercaptomethylthiolane, and SAPS(N-(4-[211At] astatophenethyl)succinimate).

One chelating agent,1,4,7,10-tetraazacyclododecane-N,N,N″,N′″-tetraacetic acid (DOTA), is ofparticular interest because of its ability to chelate a number ofdiagnostically and therapeutically important metals, such asradionuclides and radiolabels.

Conjugates of DOTA and proteins such as antibodies have been described.For example, U.S. Pat. No. 5,428,156 teaches a method for conjugatingDOTA to antibodies and antibody fragments. To make these conjugates, onecarboxylic acid group of DOTA is converted to an active ester which canreact with an amine or sulfhydryl group on the antibody or antibodyfragment. Lewis et al. (1994) Bioconjugate Chem. 5: 565-576, describes asimilar method wherein one carboxyl group of DOTA is converted to anactive ester, and the activated DOTA is mixed with an antibody, linkingthe antibody to DOTA via the epsilon-amino group of a lysine residue ofthe antibody, thereby converting one carboxyl group of DOTA to an amidemoiety.

Alternatively the chelating agent can be coupled, directly or through alinker, to an epitope tag or to a moiety that binds an epitope tag.Conjugates of DOTA and biotin have been described (see, e.g., Su (1995)J. Nucl. Med., 36 (5 Suppl):154P, which discloses the linkage of DOTA tobiotin via available amino side chain biotin derivatives such asDOTA-LC-biotin or DOTA-benzyl-4-(6-amino-caproamide)-biotin). Yau etal., WO 95/15335, disclose a method of producing nitro-benzyl-DOTAcompounds that can be conjugated to biotin. The method comprises acyclization reaction via transient projection of a hydroxy group;tosylation of an amine; deprotection of the transiently protectedhydroxy group; tosylation of the deprotected hydroxy group; andintramolecular tosylate cyclization. Wu et al. (1992) Nucl. Med. Biol.,19(2): 239-244 discloses a synthesis of macrocylic chelating agents forradiolabeling proteins with ¹¹¹IN and ⁹⁰Y. Wu et al. makes a labeledDOTA-biotin conjugate to study the stability and biodistribution ofconjugates with avidin, a model protein for studies. This conjugate wasmade using a biotin hydrazide which contained a free amino group toreact with an in situ generated activated DOTA derivative.

4) Cytotoxins.

Particularly preferred cytotoxins include Pseudomonas exotoxins,Diphtheria toxins, ricin, and abrin. Pseudomonas exotoxin and Dipthteriatoxin are most preferred.

Pseudomonas exotoxin A (PE) is an extremely active monomeric protein(molecular weight 66 kD), secreted by Pseudomonas aeruginosa, whichinhibits protein synthesis in eukaryotic cells through the inactivationof elongation factor 2 (EF-2) by catalyzing its ADP-ribosylation(catalyzing the transfer of the ADP ribosyl moiety of oxidized NAD ontoEF-2).

The toxin contains three structural domains that act in concert to causecytotoxicity. Domain Ia (amino acids 1-252) mediates cell binding.Domain II (amino acids 253-364) is responsible for translocation intothe cytosol and domain III (amino acids 400-613) mediates ADPribosylation of elongation factor 2, which inactivates the protein andcauses cell death. The function of domain Ib (amino acids 365-399)remains undefined, although a large part of it, amino acids 365-380, canbe deleted without loss of cytotoxicity. See Siegall et al. (1989) J.Biol. Chem. 264: 14256-14261.

Where the targeting molecule is fused to PE, a preferred PE molecule isone in which domain Ia (amino acids 1 through 252) is deleted and aminoacids 365 to 380 have been deleted from domain Ib. However all of domainIb and a portion of domain II (amino acids 350 to 394) can be deleted,particularly if the deleted sequences are replaced with a linkingpeptide such as GGGGS (SEQ ID NO:11).

In addition, the PE molecules can be further modified usingsite-directed mutagenesis or other techniques known in the art, to alterthe molecule for a particular desired application. Means to alter the PEmolecule in a manner that does not substantially affect the functionaladvantages provided by the PE molecules described here can also be usedand such resulting molecules are intended to be covered herein.

For maximum cytotoxic properties of a preferred PE molecule, severalmodifications to the molecule are recommended. An appropriate carboxylterminal sequence to the recombinant molecule is preferred totranslocate the molecule into the cytosol of target cells. Amino acidsequences which have been found to be effective include, REDLK (SEQ IDNO:23) (as in native PE), REDL (SEQ ID NO:24), RDEL (SEQ ID NO:25), orKDEL (SEQ ID NO:26), repeats of those, or other sequences that functionto maintain or recycle proteins into the endoplasmic reticulum, referredto here as “endoplasmic retention sequences”. See, for example,Chaudhary et al. (1991) Proc. Natl. Acad. Sci. USA 87:308-312 andSeetharam et al, J. Biol. Chem. 266: 17376-17381. Preferred forms of PEcomprise the PE molecule designated PE38QQR. (Debinski et al. Bioconj.Chem., 5: 40 (1994)), and PE4E (see, e.g., Chaudhary et al. (1995) J.Biol. Chem., 265: 16306).

Methods of cloning genes encoding PE fused to various ligands are wellknown to those of skill in the art (see, e.g., Siegall et al. (1989)FASEB J., 3: 2647-2652; and Chaudhary et al. (1987) Proc. Natl. Acad.Sci. USA, 84: 4538-4542).

Like PE, diphtheria toxin (DT) kills cells by ADP-ribosylatingelongation factor 2 thereby inhibiting protein synthesis. Diphtheriatoxin, however, is divided into two chains, A and B, linked by adisulfide bridge. In contrast to PE, chain B of DT, which is on thecarboxyl end, is responsible for receptor binding and chain A, which ispresent on the amino end, contains the enzymatic activity (Uchida et al.(1972) Science, 175: 901-903; Uchida et al. (1973) J. Biol. Chem., 248:3838-3844).

In a preferred embodiment, the targeting molecule-Diphtheria toxinfusion proteins of this invention have the native receptor-bindingdomain removed by truncation of the Diphtheria toxin B chain.Particularly preferred is DT388, a DT in which the carboxyl terminalsequence beginning at residue 389 is removed. Chaudhary et al. (1991)Bioch. Biophys. Res. Comm., 180: 545-551. Like the PE chimericcytotoxins, the DT molecules may be chemically conjugated to theantibody, but, in certain preferred embodiments, the targeting moleculewill be fused to the Diphtheria toxin by recombinant means (see, e.g.,Williams et al. (1990) J. Biol. Chem. 265: 11885-11889).

5) Other Therapeutic Moieties.

Other suitable effector molecules include pharmacological agents orencapsulation systems containing various pharmacological agents. Thus,the targeting molecule of the chimeric molecule may be attached directlyto a drug that is to be delivered directly to the tumor. Such drugs arewell known to those of skill in the art and include, but are not limitedto, doxirubicin, vinblastine, genistein, an antisense molecule, and thelike.

Alternatively, the effector molecule may be an encapsulation system,such as a viral capsid, a liposome, or micelle that contains atherapeutic composition such as a drug, a nucleic acid (e.g. anantisense nucleic acid), or another therapeutic moiety that ispreferably shielded from direct exposure to the circulatory system.Means of preparing liposomes attached to antibodies are well known tothose of skill in the art. See, for example, U.S. Pat. No. 4,957,735,Connor et al. (1985) Pharm. Ther., 28: 341-365.

C) Attachment of the Targeting Molecule to the Effector Molecule.

One of skill will appreciate that the a bispecific and/or polyspecificantibody of this invention and the effector moieties can typically bejoined together in any order. Thus, for example, where the targetingmolecule is a single chain protein the effector molecule may be joinedto either the amino or carboxy termini of the targeting molecule. Theeffector can also be joined to an internal region of the a bispecificand/or polyspecific antibody, or conversely. Similarly, the a bispecificand/or polyspecific antibody can be joined to an internal location or aterminus of the effector molecule. In any case, attachment points areselected that do not interfere with the respective activities of the abispecific and/or polyspecific antibody or the effector.

The bispecific and/or polyspecific antibody and the effector moleculecan be attached by any of a number of means well known to those of skillin the art. Typically the effector molecule is conjugated, eitherdirectly or through a linker (spacer), to the bispecific antibody.However, where both the effector molecule and the bispecific antibodyare both polypeptides it is preferable to recombinantly express thechimeric molecule as a single-chain fusion protein.

1) Conjugation of the Effector Molecule to the Targeting Molecule.

In one embodiment, the a bispecific and/or polyspecific antibody ischemically conjugated to the effector molecule (e.g., a cytotoxin, alabel, a ligand, a drug, an antibody, a liposome, etc.). Means ofchemically conjugating molecules are well known to those of skill.

The procedure for attaching an agent to an antibody or other polypeptidetargeting molecule will vary according to the chemical structure of theagent. Polypeptides typically contain variety of functional groups;e.g., carboxylic acid (COOH) or free amine (—NH₂) groups, which areavailable for reaction with a suitable functional group on an effectormolecule to bind the effector thereto.

Alternatively, the bispecific antibody and/or effector molecule can bederivatized to expose or attach additional reactive functional groups.The derivatization can involve attachment of any of a number of linkermolecules such as those available from Pierce Chemical Company, RockfordIll.

A “linker”, as used herein, is a molecule that is used to join thetargeting molecule to the effector molecule. The linker is capable offorming covalent bonds to both the targeting molecule and to theeffector molecule. Suitable linkers are well known to those of skill inthe art and include, but are not limited to, straight or branched-chaincarbon linkers, heterocyclic carbon linkers, or peptide linkers. Wherethe a bispecific and/or polyspecific antibody and the effector moleculeare polypeptides, the linkers can be joined to the constituent aminoacids through their side groups (e.g., through a disulfide linkage tocysteine). However, in a preferred embodiment, the linkers will bejoined to the alpha carbon amino and carboxyl groups of the terminalamino acids.

A bifunctional linker having one functional group reactive with a groupon a particular agent, and another group reactive with an antibody, canbe used to form the desired immunoconjugate. Alternatively,derivatization can involve chemical treatment of the a bispecific and/orpolyspecific antibody, e.g., glycol cleavage of a sugar moiety of aglycoprotein antibody with periodate to generate free aldehyde groups.The free aldehyde groups on the antibody can be reacted with free amineor hydrazine groups on an agent to bind the agent thereto. (See U.S.Pat. No. 4,671,958). Procedures for generation of free sulfhydryl groupson polypeptide, such as antibodies or antibody fragments, are also known(See U.S. Pat. No. 4,659,839).

Many procedures and linker molecules for attachment of various compoundsincluding radionuclide metal chelates, toxins and drugs to proteins suchas antibodies are known (see, e.g., European Patent Application No.188,256; U.S. Pat. Nos. 4,671,958, 4,659,839, 4,414,148, 4,699,784;4,680,338; 4,569,789; and 4,589,071; and Borlinghaus et al. (1987)Cancer Res. 47: 4071-4075). In particular, production of variousimmunotoxins is well-known within the art and can be found, for examplein “Monoclonal Antibody-Toxin Conjugates: Aiming the Magic Bullet,”Thorpe et al., Monoclonal Antibodies in Clinical Medicine, AcademicPress, pp. 168-190 (1982), Waldmann (1991) Science, 252: 1657, U.S. Pat.Nos. 4,545,985 and 4,894,443.

In some circumstances, it is desirable to free the effector moleculefrom the a bispecific and/or polyspecific antibody when the chimericmoiety has reached its target site. Therefore, chimeric conjugatescomprising linkages that are cleavable in the vicinity of the targetsite can be used when the effector is to be released at the target site.Cleaving of the linkage to release the agent from the antibody may beprompted by enzymatic activity or conditions to which theimmunoconjugate is subjected either inside the target cell or in thevicinity of the target site. When the target site is a tumor, a linkerwhich is cleavable under conditions present at the tumor site (e.g. whenexposed to tumor-associated enzymes or acidic pH) may be used.

A number of different cleavable linkers are known to those of skill inthe art. See U.S. Pat. Nos. 4,618,492; 4,542,225, and 4,625,014. Themechanisms for release of an agent from these linker groups include, forexample, irradiation of a photolabile bond and acid-catalyzedhydrolysis. U.S. Pat. No. 4,671,958, for example, includes a descriptionof immunoconjugates comprising linkers which are cleaved at the targetsite in vivo by the proteolytic enzymes of the patient's complementsystem. In view of the large number of methods that have been reportedfor attaching a variety of radiodiagnostic compounds, radiotherapeuticcompounds, drugs, toxins, and other agents to antibodies one skilled inthe art will be able to determine a suitable method for attaching agiven agent to an antibody or other polypeptide.

2 Conjugation of Chelates.

In certain preferred embodiments, the effector comprises a chelate thatis attached to an antibody or to an epitope tag. The a bispecific and/orpolyspecific antibody bears a corresponding epitope tag or antibody sothat simple contacting of the a bispecific and/or polyspecific antibodyto the chelate results in attachment of the antibody to the effector.The combining step can be performed after the moiety is used(pretargeting strategy) or the target tissue can be bound to the abispecific and/or polyspecific antibody before the chelate is delivered.Methods of producing chelates suitable for coupling to various targetingmoieties are well known to those of skill in the art (see, e.g., U.S.Pat. Nos. 6,190,923, 6,187,285, 6,183,721, 6,177,562, 6,159,445,6,153,775, 6,149,890, 6,143,276, 6,143,274, 6,139,819, 6,132,764,6,123,923, 6,123,921, 6,120,768, 6,120,751, 6,117,412, 6,106,866,6,096,290, 6,093,382, 6,090,800, 6,090,408, 6,088,613, 6,077,499,6,075,010, 6,071,494, 6,071,490, 6,060,040, 6,056,939, 6,051,207,6,048,979, 6,045,821, 6,045,775, 6,030,840, 6,028,066, 6,022,966,6,022,523, 6,022,522, 6,017,522, 6,015,897, 6,010,682, 6,010,681,6,004,533, and 6,001,329).

3) Production of Fusion Proteins.

Where the a bispecific and/or polyspecific antibody and/or the effectormolecule are both single chain proteins and relatively short (i.e., lessthan about 50 amino acids) they can be synthesized using standardchemical peptide synthesis techniques. Where both components arerelatively short the chimeric moiet6y can be synthesized as a singlecontiguous polypeptide. Alternatively the a bispecific and/orpolyspecific antibody and the effector molecule may be synthesizedseparately and then fused by condensation of the amino terminus of onemolecule with the carboxyl terminus of the other molecule therebyforming a peptide bond. Alternatively, the a bispecific and/orpolyspecific antibody and effector molecules may each be condensed withone end of a peptide spacer molecule thereby forming a contiguous fusionprotein.

Solid phase synthesis in which the C-terminal amino acid of the sequenceis attached to an insoluble support followed by sequential addition ofthe remaining amino acids in the sequence is the preferred method forthe chemical synthesis of the polypeptides of this invention. Techniquesfor solid phase synthesis are described by Barany and Merrifield,Solid-Phase Peptide Synthesis; pp. 3-284 in The Peptides: Analysis,Synthesis, Biology. Vol. 2: Special Methods in Peptide Synthesis, PartA., Merrifield, et al. J. Am. Chem. Soc., 85: 2149-2156 (1963), andStewart et al., Solid Phase Peptide Synthesis, 2nd ed. Pierce Chem. Co.,Rockford, Ill. (1984).

In a preferred embodiment, the where the a bispecific and/orpolyspecific antibody is a single chain polypeptide and the effector isa polypeptide, chimeric fusion proteins of the present invention aresynthesized using recombinant DNA methodology. Generally this involvescreating a DNA sequence that encodes the fusion protein, placing the DNAin an expression cassette under the control of a particular promoter,expressing the protein in a host, isolating the expressed protein and,if required, renaturing the protein.

DNA encoding the fusion proteins (e.g. ALM-PE38QQR) of this inventionmay be prepared by any suitable method, including, for example, cloningand restriction of appropriate sequences or direct chemical synthesis bymethods such as the phosphotriester method of Narang et al. (1979) Meth.Enzymol. 68: 90-99; the phosphodiester method of Brown et al. (1979)Meth. Enzymol. 68: 109-151; the diethylphosphoramidite method ofBeaucage et al. (1981) Tetra. Lett., 22: 1859-1862; and the solidsupport method of U.S. Pat. No. 4,458,066.

Chemical synthesis produces a single stranded oligonucleotide. This maybe converted into double stranded DNA by hybridization with acomplementary sequence, or by polymerization with a DNA polymerase usingthe single strand as a template. One of skill would recognize that whilechemical synthesis of DNA is limited to sequences of about 100 bases,longer sequences can be obtained by the ligation of shorter sequences.

Alternatively, subsequences can be cloned and the appropriatesubsequences cleaved using appropriate restriction enzymes. Thefragments can then be ligated to produce the desired DNA sequence.

In a preferred embodiment, DNA encoding fusion proteins of the presentinvention may be cloned using DNA amplification methods such aspolymerase chain reaction (PCR). Thus, for example, the nucleic acidencoding a bispecific and/or polyspecific antibody is PCR amplified,using a sense primer containing the restriction site for NdeI and anantisense primer containing the restriction site for HindIII. Thisproduces a nucleic acid encoding the a bispecific and/or polyspecificantibody sequence and having terminal restriction sites. A PE38QQRfragment can be cut out of the plasmid pWDMH4-38QQR or plasmidpSGC242FdN1 described by Debinski et al. (1994) Int. J. Cancer, 58:744-748. Ligation of the a bispecific and/or polyspecific antibody andPE38QQR sequences and insertion into a vector produces a vector encodingthe bispecific and/or polyspecific antibody joined to the amino terminusof PE38QQR (position 253 of PE). The two molecules are joined by a threeamino acid junction consisting of glutamic acid, alanine, andphenylalanine introduced by the restriction site.

While the two molecules are preferably essentially directly joinedtogether, one of skill will appreciate that the molecules may beseparated by a peptide spacer consisting of one or more amino acids.Generally the spacer will have no specific biological activity otherthan to join the proteins or to preserve some minimum distance or otherspatial relationship between them. However, the constituent amino acidsof the spacer can be selected to influence some property of the moleculesuch as the folding, net charge, or hydrophobicity.

The nucleic acid sequences encoding the fusion proteins can be expressedin a variety of host cells, including E. coli, other bacterial hosts,yeast, and various higher eukaryotic cells such as the COS, CHO and HeLacells lines and myeloma cell lines. The recombinant protein gene will beoperably linked to appropriate expression control sequences for eachhost. For E. coli this includes a promoter such as the T7, trp, orlambda promoters, a ribosome binding site and preferably a transcriptiontermination signal. For eukaryotic cells, the control sequences willinclude a promoter and preferably an enhancer derived fromimmunoglobulin genes, SV40, cytomegalovirus, etc., and a polyadenylationsequence, and may include splice donor and acceptor sequences.

The plasmids of the invention can be transferred into the chosen hostcell by well-known methods such as calcium chloride transformation forE. coli and calcium phosphate treatment or electroporation for mammaliancells. Cells transformed by the plasmids can be selected by resistanceto antibiotics conferred by genes contained on the plasmids, such as theamp, gpt, neo and hyg genes.

Once expressed, the recombinant fusion proteins can be purifiedaccording to standard procedures of the art, including ammonium sulfateprecipitation, affinity columns, column chromatography, gelelectrophoresis and the like (see, generally, R. Scopes (1982) ProteinPurification, Springer-Verlag, N.Y.; Deutscher (1990) Methods inEnzymology Vol. 182: Guide to Protein Purification., Academic Press,Inc. N.Y.). Substantially pure compositions of at least about 90 to 95%homogeneity are preferred, and 98 to 99% or more homogeneity are mostpreferred for pharmaceutical uses. Once purified, partially or tohomogeneity as desired, the polypeptides may then be usedtherapeutically.

One of skill in the art would recognize that after chemical synthesis,biological expression, or purification, the EGFR polypeptide targetedfusion protein can possess a conformation substantially different thanthe native conformations of the constituent polypeptides. In this case,it may be necessary to denature and reduce the polypeptide and then tocause the polypeptide to re-fold into the preferred conformation.Methods of reducing and denaturing proteins and inducing re-folding arewell known to those of skill in the art (See, Debinski et al. (1993) J.Biol. Chem., 268: 14065-14070; Kreitman and Pastan (1993) Bioconjug.Chem., 4: 581-585; and Buchner, et al. (1992) Anal. Biochem., 205:263-270).

One of skill would recognize that modifications can be made to thefusion proteins without diminishing their biological activity. Somemodifications may be made to facilitate the cloning, expression, orincorporation of the targeting molecule into a fusion protein. Suchmodifications are well known to those of skill in the art and include,for example, a methionine added at the amino terminus to provide aninitiation site, or additional amino acids placed on either terminus tocreate conveniently located restriction sites or termination codons.

IV. Uses of Bispecific Antibody Molecules and/or Chimeric Moieties:

Bispecific antibodies having affinity for two distinct antigens havebroad applications in therapy and diagnosis. Specifically, thebs-antibody molecules of the invention (e.g., bs-scFv)., can be used:(1) to directly alter the growth of tumors that overexpress members ofthe EGFR protein family; (2) in combination with other cytotoxic agents(e.g., chemotherapeutic agents, external beam radiation, targetedradioisotopes, and other antibodies or signal transduction inhibitors);and (3) to recruit a variety of different cytotoxic agents or effectorcells directly to targeted tumor cells that express members of the EGFRprotein family.

Targeting cytotoxic agents or effector cells to specific tumor cellsutilizing the bs-scFv antibody molecules of the invention provides addedtumor-directed specificity due to the increased expression of thesetargets on tumor cells relative to normal tissue. In addition, thebispecific antibodies can bind to multiple receptors or receptorcomponents, thereby cross-linking receptors or receptor componentsproducing a cytotoxic and/or cytostatic effect. Antibody-based agentsthat only bind to one target on normal tissue will typically notcrosslink the receptors and trigger cytotoxic results.

In addition, monospecific antibodies typically show lower avidity to thetarget cell. In contrast, the bispecific antibodies of this inventionshow higher avidity to the target cell(s) which helps stabilize theantibody/target complex and provide long-term association of theantibody with the cell, thus providing added specificity for the agenton tumor cells that overexpress both targets.

In addition, the binding of antibodies to the members of the EGFRprotein family often triggers the internalization of these proteins,making these antibodies effective platforms for the delivery of toxins,drugs, radioisotopes or other cytotoxic agents. ALM mediates a reductionin the quantity of HER2/neu and HER3 on the surface of tumor cells,suggesting a similar internalization mechanism. Therefore, thecombination of these bs-scFv molecules with cytotoxic or other agents(effectors), e.g. in a chimeric moiety, will result in effectivedelivery to cells that overexpress both targets, thus increasing thespecificity and efficacy of the therapy. By incorporating additionalsequences (e.g., Fc receptor targeting arms) that interact with effectorcells, a similar increase in targeting specificity can also beincorporated into effector cell-based treatment strategies.

The bispecific antibody molecules of the invention can also be used ingene therapy for direct targeting and internalization of nucleic acidsencoding therapeutic agents (e.g. pseudomonas exotoxin, diphtheriatoxin, various tumor suppressor genes, various labels, etc.), Inaddition, the bispecific antibodies can be conjugated, e.g. via achelate to cytotoxic radioactive moieties (e.g. ²¹¹At), to radiationenhancing agents, and to various detectable labels (e.g. radio opaquelabels). In addition, the bispecific antibody molecules can be coupledto lipids, liposomes, dendrimers, and the like. The lipids, liposomesand dendrimers can combine with and/or encapsulate various therapeuticmoieties (e.g. anticancer drugs including, but not limited to,alkylating agents such as busulfan, chlorambuicl, cis-platinum,cyanomorpholinodoxorubicin, etc., antimitotic agents such asallocolchicine, cohchicine, taxol, vinblastine, vincristine, and thelike, topoisomerase I inhibitors such as camptothecin,aminocamptothecin, and the like, topoisomerase II inhibitors such asdoxorubicin, amonafide, daunorubicin, deoxydoxorubicin, mitoxantrone,and the like, RNA/DNA antimetabolites such as acivicin, ftorafur,methotrexate, trimetrexate, and the like; DNA antimetabolites such as 2′deoxy-5-fluorouridine, cyclocytidine, guanazole, and the like). Lipids,liposomes and dendrimers can also complex with protein therapeutics,nucleic acids encoding, e.g. therapeutic moieties, and the like.

When used as a targeting component of a chimeric moiety, as describedabove, the bispecific and/or polyspecific antibodies of this inventionpreferentially target/deliver the associated effector to the targetcell(s) expressing the target EGFR proteins. By increasing theassociation (e.g. duration of contact or amount of contact) of theeffector with the cell (in contact or close proximity), the antibodiesof this invention increase the likelihood of the effector internalizinginto the cell and/or exerting its characteristic activity on that cell.

Thus, for example, bispecific or polyspecific antibody targetedliposomes or other therapeutic vesicles (liposomes, viruses etc.) showincreased exposure (duration/concentration) to target tumors. In anexemplary embodiment, liposomes can be studded by the bs-scFv antibodymolecules of the invention to facilitate tumor specific targeting.Anti-cancer agents such as chemotherapeutic agents, antibodies,antisense molecules and/or radioisotopes may be encapsulated inliposomes so modified.

In another embodiment, the bispecific or poylyspecific antibody (e.g.,bs-scFv antibody) molecules can be used to direct gene therapy vectors,including but not limited to modified viruses, to cells that expressboth target antigens. Viruses can also be utilized to deliver the genesfor these bs-scFv antibody molecules to tumor cells where they could beproduced and secreted into the cellular microenvironment or, through theaddition of additional intracellular targeting sequences, they could beturned into intrabodies that localize to specific cellular compartmentsand knockout the expression of their targets.

In addition, the bispecific or poylyspecific antibody (e.g., bs-scFvantibody) molecules of the invention can be used to advantage to detectaberrant expression of members of the EGFR protein family. Suchdetection can lead to early diagnosis of cancers associated withaberrant tumor growth facilitated by these cell surface proteins. Ingeneral, the detection of immunocomplex formation is well known in theart and can be achieved through the application of numerous approaches.These methods are generally based upon the detection of a label ormarker, such as any radioactive, fluorescent, biological or enzymatictags or labels of standard use in the art. U.S. patents concerning theuse of such labels include U.S. Pat. Nos. 3,817,837; 3,850,752;3,939,350; 3,996,345; 4,277,437; 4,275,149 and 4,366,241. Of course, onemay find additional advantages through the use of a secondary bindingligand such as a second antibody or a biotin/avidin ligand bindingarrangement, as is known in the art.

V. Administration of Bs-scFv Antibody Molecules:

A) Pharmaceutical Formulations.

Bispecific antibodies or bs-scFv antibody molecules or chimericmoieties, as described herein, include bulk drug compositions useful inthe manufacture of non-pharmaceutical compositions (e.g., impure ornon-sterile compositions), and pharmaceutical compositions (i.e.,compositions that are suitable for administration to a subject orpatient (i.e., human or non-human subject) that can be used directlyand/or in the preparation of unit dosage forms. In certain embodiments,such compositions comprise a therapeutically effective amount of one ormore therapeutic agents (e.g. bispecific and/or polyspecific antibodies,and/or chimeric moieties comprising such antibodies) and apharmaceutically acceptable carrier.

As indicated above, the agents of this invention can be used in a widevariety of contexts including, but not limited to the detection and/orimaging of tumors or cancer cells, inhibition of tumor growth and/orcancer cell growth and/or proliferation, and the like. One or morebispecific antibodies, and/or functionalized bispecific antibodies,and/or chimeric moieties of this invention can be administered byinjection, that is, intravenously, intramuscularly, intracutaneously,subcutaneously, intraduodenally, or intraperitoneally. Also, in certainembodiments, the compounds can be administered by inhalation, forexample, intranasally. Additionally, certain compounds can beadministered orally, or transdermally.

In a specific embodiment, the term “pharmaceutically acceptable” meansapproved by a regulatory agency of the Federal or a state government orlisted in the U.S. Pharmacopeia or other generally recognizedpharmacopeia for use in animals, and more particularly in humans, orsuitable for administration to an animal or human. The term “carrier” orrefers to a diluent, adjuvant (e.g., Freund's adjuvant (complete andincomplete)), excipient, or vehicle with which the therapeutic isadministered. Such pharmaceutical carriers can be sterile liquids, suchas water and oils, including those of petroleum, animal, vegetable orsynthetic origin, such as peanut oil, soybean oil, mineral oil, sesameoil and the like. Water is a preferred carrier when the pharmaceuticalcomposition is administered intravenously. Saline solutions and aqueousdextrose and glycerol solutions can also be employed as liquid carriers,particularly for injectable solutions. Suitable pharmaceuticalexcipients include starch, glucose, lactose, sucrose, gelatin, malt,rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate,talc, sodium chloride, dried skim milk, glycerol, propylene, glycol,water, ethanol and the like. The composition, if desired, can alsocontain minor amounts of wetting or emulsifying agents, or pH bufferingagents. These compositions can take the form of solutions, suspensions,emulsion, tablets, pills, capsules, powders, sustained-releaseformulations and the like.

Generally, the ingredients of the compositions of the invention aresupplied either separately or mixed together in unit dosage form, forexample, as a dry lyophilized powder or water free concentrate in ahermetically sealed container such as an ampoule or sachette indicatingthe quantity of active agent. Where the composition is to beadministered by infusion, it can be dispensed with an infusion bottlecontaining sterile pharmaceutical grade water or saline. Where thecomposition is administered by injection, an ampoule of sterile waterfor injection or saline can be provided so that the ingredients may bemixed prior to administration.

The compositions of the invention can be provided as neutral or saltforms. Pharmaceutically acceptable salts include those formed withanions such as those derived from hydrochloric, phosphoric, acetic,oxalic, tartaric acids, etc., and those formed with cations such asthose derived from sodium, potassium, ammonium, calcium, ferrichydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol,histidine, procaine, etc.

Pharmaceutical compositions comprising the bispecific antibodies, and/orfunctionalized bispecific antibodies, and/or chimeric moieties of thisinvention can be manufactured by means of conventional mixing,dissolving, granulating, dragee-making, levigating, emulsifying,encapsulating, entrapping or lyophilizing processes. Pharmaceuticalcompositions may be formulated in conventional manner using one or morephysiologically acceptable carriers, diluents, excipients or auxiliariesthat facilitate processing of the molecules into preparations that canbe used pharmaceutically. Proper formulation is dependent upon the routeof administration chosen.

For topical or transdermal administration, the bispecific antibodies,and/or functionalized bispecific antibodies, and/or chimeric moieties ofthis invention can be formulated as solutions, gels, ointments, creams,lotion, emulsion, suspensions, etc. as are well-known in the art.Systemic formulations include those designed for administration byinjection, e.g. subcutaneous, intravenous, intramuscular, intrathecal orintraperitoneal injection, as well as those designed for transdermal,transmucosal, inhalation, oral or pulmonary administration.

For injection, the bispecific antibodies, and/or functionalizedbispecific antibodies, and/or chimeric moieties of this invention can beformulated in aqueous solutions, preferably in physiologicallycompatible buffers such as Hanks's solution, Ringer's solution, orphysiological saline buffer. The solution can contain formulatory agentssuch as suspending, stabilizing and/or dispersing agents. Alternatively,compositions comprising the iron chelating agent(s) can be in powderform for constitution with a suitable vehicle, e.g., sterilepyrogen-free water, before use.

For transmucosal administration, penetrants appropriate to the barrierto be permeated are used in the formulation. Such penetrants aregenerally known in the art.

For oral administration, the bispecific antibodies, and/orfunctionalized bispecific antibodies, and/or chimeric moieties of thisinvention can be readily formulated by combining the agent(s) withpharmaceutically acceptable carriers well known in the art. Suchcarriers enable the agent(s) to be formulated as tablets, pills,dragees, capsules, liquids, gels, syrups, slurries, suspensions and thelike, for oral ingestion by a patient to be treated. For oral solidformulations such as, for example, powders, capsules and tablets,suitable excipients include fillers such as sugars, e.g. lactose,sucrose, mannitol and sorbitol; cellulose preparations such as maizestarch, wheat starch, rice starch, potato starch, gelatin, gumtragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodiumcarboxymethylcellulose, and/or polyvinylpyrrolidone (PVP); granulatingagents; and binding agents. If desired, disintegrating agents may beadded, such as the cross-linked polyvinylpyrrolidone, agar, or alginicacid or a salt thereof such as sodium alginate.

If desired, solid dosage forms may be sugar-coated or enteric-coatedusing standard techniques.

For oral liquid preparations such as, for example, suspensions, elixirsand solutions, suitable carriers, excipients or diluents include water,glycols, oils, alcohols, etc. Additionally, flavoring agents,preservatives, coloring agents and the like can be added.

For buccal administration, the iron chelating agent(s) can take the formof tablets, lozenges, etc. formulated in conventional manner.

For administration by inhalation, bispecific antibodies, and/orfunctionalized bispecific antibodies, and/or chimeric moieties of thisinvention are conveniently delivered in the form of an aerosol sprayfrom pressurized packs or a nebulizer, with the use of a suitablepropellant, e.g., dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In thecase of a pressurized aerosol, the dosage unit may be determined byproviding a valve to deliver a metered amount. Capsules and cartridgesof gelatin for use in an inhaler or insufflator may be formulatedcontaining a powder mix of the iron chelating agent(s) and a suitablepowder base such as lactose or starch.

The bispecific antibodies, and/or functionalized bispecific antibodies,and/or chimeric moieties of this invention (can also be formulated inrectal or vaginal compositions such as suppositories or retentionenemas, e.g, containing conventional suppository bases such as cocoabutter or other glycerides.

In addition to the formulations described previously, the bispecificantibodies, and/or functionalized bispecific antibodies, and/or chimericmoieties of this invention can also be formulated as a depotpreparation. Such long acting formulations may be administered byimplantation (for example subcutaneously or intramuscularly) or byintramuscular injection. Thus, for example, the agent(s) of thisinvention can be formulated with suitable polymeric or hydrophobicmaterials (for example as an emulsion in an acceptable oil) or ionexchange resins, or as sparingly soluble derivatives, for example, as asparingly soluble salt.

Other pharmaceutical delivery systems can also be employed. Liposomesand emulsions are well known examples of delivery vehicles that may beused to deliver the bispecific antibodies, and/or functionalizedbispecific antibodies, and/or chimeric moieties of this invention.Certain organic solvents such as dimethylsulfoxide also may be employed,although usually at the cost of greater toxicity. Additionally, thebispecific antibodies, and/or functionalized bispecific antibodies,and/or chimeric moieties of this invention can be delivered using asustained-release system, such as semipermeable matrices of solidpolymers containing the therapeutic agent. Various sustained-releasematerials have been established and are well known by those skilled inthe art. Sustained-release capsules may, depending on their chemicalnature, can release the active agent(s) for a few days, a few weeks, orup to over 100 days. Depending on the chemical nature and the biologicalstability of the agent(s) additional strategies for stabilization can beemployed.

As the bispecific antibodies, and/or functionalized bispecificantibodies, and/or chimeric moieties of this invention may containcharged side chains or termini, they can be included in any of theabove-described formulations as the free acids or bases or aspharmaceutically acceptable salts. Pharmaceutically acceptable salts arethose salts which substantially retain the biological activity of thefree bases and which are prepared by reaction with inorganic acids.Pharmaceutical salts tend to be more soluble in aqueous and other proticsolvents than are the corresponding free base forms.

B) Effective Dosages.

The bispecific antibodies, and/or functionalized bispecific antibodies,and/or chimeric moieties of this invention will generally be used in anamount effective to achieve the intended purpose (e.g. to image a tumoror cancer cell, to inhibit growth and/or proliferation of cancer cells,etc.). In certain preferred embodiments, the bispecific antibodies,and/or functionalized bispecific antibodies, and/or chimeric moietiesutilized in the methods of this invention are administered at a dosethat is effective to partially or fully inhibit cancer cellproliferation and/or growth, or to enable visualization of a cancer cellor tumor characterized by overexpression of an EGFR family protein. Incertain embodiments, dosages are selected that inhibit cancer cellgrowth and/or proliferation at the 90%, more preferably at the 95%, andmost preferably at the 98% or 99% confidence level. Preferred effectiveamounts are those that reduce or prevent tumor growth or that facilitatecancer cell detection and/or visualization. With respect to inhibitorsof cell growth and proliferation, the compounds can also be usedprophalactically at the same dose levels.

Typically, bispecific antibodies, and/or functionalized bispecificantibodies, and/or chimeric moieties of this invention, orpharmaceutical compositions thereof, are administered or applied in atherapeutically effective amount. A therapeutically effective amount isan amount effective to reduce or prevent the onset or progression (e.g,growth and/or proliferation) of a cancer cell and/or a tumor.Determination of a therapeutically effective amount is well within thecapabilities of those skilled in the art, especially in light of thedetailed disclosure provided herein.

For systemic administration, a therapeutically effective dose can beestimated initially from in vitro assays. For example, a dose can beformulated in animal models to achieve a circulating concentration rangethat includes the IC₅₀ as determined in cell culture. Such informationcan be used to more accurately determine useful doses in humans.

Initial dosages can also be estimated from in vivo data, e.g., animalmodels, using techniques that are well known in the art. One skilled inthe art could readily optimize administration to humans based on animaldata.

Dosage amount and interval can be adjusted individually to provideplasma levels of the inhibitors which are sufficient to maintaintherapeutic effect.

Dosages for typical therapeutics are known to those of skill in the art.Moreover, such dosages are typically advisorial in nature and may beadjusted depending on the particular therapeutic context, patienttolerance, etc. Single or multiple administrations of the compositionsmay be administered depending on the dosage and frequency as requiredand tolerated by the patient.

In certain embodiments, an initial dosage of about 1 μg, preferably fromabout 1 mg to about 1000 mg per kilogram daily will be effective. Adaily dose range of about 5 to about 75 mg is preferred. The dosages,however, can be varied depending upon the requirements of the patient,the severity of the condition being treated, and the compound beingemployed. Determination of the proper dosage for a particular situationis within the skill of the art. Generally, treatment is initiated withsmaller dosages that are less than the optimum dose of the compound.Thereafter, the dosage is increased by small increments until theoptimum effect under the circumstance is reached. For convenience, thetotal daily dosage can be divided and administered in portions duringthe day if desired. Typical dosages will be from about 0.1 to about 500mg/kg, and ideally about 25 to about 250 mg/kg.

In cases of local administration or selective uptake, the effectivelocal concentration of the bispecific antibodies and/or chimericmolecules may not be related to plasma concentration. One skilled in theart will be able to optimize therapeutically effective local dosageswithout undue experimentation. The amount of antibody and/or chimericmoiety will, of course, be dependent on the subject being treated, onthe subject's weight, the severity of the affliction, the manner ofadministration and the judgment of the prescribing physician.

The therapy can be repeated intermittently. In certain embodiments, thepharmaceutical preparation comprising the bispecific antibody moleculescam be administered at appropriate intervals, for example, at leasttwice a day or more until the pathological symptoms are reduced oralleviated, after which the dosage may be reduced to a maintenancelevel. The appropriate interval in a particular case would normallydepend on the condition of the patient. The therapy can be providedalone or in combination with other drugs, and/or radiotherapy, and/orsurgical procedures.

C) Toxicity.

Preferably, a therapeutically effective dose of bispecific antibodies,and/or functionalized bispecific antibodies, and/or chimeric moieties ofthis invention described herein will provide therapeutic benefit withoutcausing substantial toxicity.

Toxicity of the agents described herein can be determined by standardpharmaceutical procedures in cell cultures or experimental animals,e.g., by determining the LD₅₀ (the dose lethal to 50% of the population)or the LD₁₀₀ (the dose lethal to 100% of the population). The dose ratiobetween toxic and therapeutic effect is the therapeutic index. Agentsthat exhibit high therapeutic indices are preferred. Data obtained fromcell culture assays and animal studies can be used in formulating adosage range that is not toxic for use in human. The dosage of thebispecific antibodies, and/or functionalized bispecific antibodies,and/or chimeric moieties of this invention preferably lie within a rangeof circulating concentrations that include the effective dose withlittle or no toxicity. The dosage can vary within this range dependingupon the dosage form employed and the route of administration utilized.The exact formulation, route of administration and dosage can be chosenby the individual physician in view of the patient's condition (see,e.g., Fingl et al. (1975) In: The Pharmacological Basis of Therapeutics,Ch.1, p. 1).

VI. Kits.

The present invention further encompasses kits for use in detectingcells expressing or overexpressing members of the EGFR protein family invivo, and/or in biological samples. Kits are also provided for ininhibiting the growth and/or proliferation of cells expressing oroverexpressing members of the Epidermal Growth Factor Family (e.g.cancer cells).

In certain embodiments, the kits comprise one or more bispecific and/orpolyspecific antibodies of this invention specific for at least twoepitopes on members of the EGFR protein family. In certain preferredembodiments, the antibodies are bispecific scFv antibodies. Depending onuse, the antibodies can be functionalized with linkers and/or chelatorsfor coupling to an effector (e.g. a radioactive moiety, a liposome, acytoxin, another antibody, etc.) as described herein.

In certain embodiments, the kits can comprise the, e.g. bs-scFv antibodymolecules of the invention specific for members of the EGFR proteinfamily as well as buffers and other compositions to be used fordetection of the bs-scFv antibody molecules.

The kits can also include instructional materials teaching the use ofthe antibodies for detecting, e.g. cancer cells, and/or teaching thecombination of the antibodies with functionalizing reagents or teachingthe use of functionalized antibodies for imaging and/or therapeuticapplications. In certain embodiments, the antibody is providedfunctionalized with a linker and/or a chelator (in one container) alongwith one or more effectors, e.g. cytotoxins, radioactive labels (in asecond container) such that the two components can be separatelyadministered (e.g. in pre-targeting approaches) or such that the twocomponents can be administered shortly before use.

Certain instructional materials will provide recommended dosage regimen,counter indications, and the like. While the instructional materialstypically comprise written or printed materials they are not limited tosuch. Any medium capable of storing such instructions and communicatingthem to an end user is contemplated by this invention. Such mediainclude, but are not limited to electronic storage media (e.g., magneticdiscs, tapes, cartridges, chips), optical media (e.g., CD ROM), and thelike. Such media may include addresses to internet sites that providesuch.

EXAMPLES

The following examples are offered to illustrate, but not to limit theclaimed invention.

Example 1 Preparation of Bs-Scfv Antibody Molecules

Overexpression of EGFR and HER2/neu has been correlated with a poorprognosis in many solid tumors. Antibodies that perturb signalingthrough these receptors, such as Herceptin⁷ (anti-HER2) and C225(anti-EGFR), have demonstrated significant utility in the treatment ofcancer. Signal transduction through members of the EGFR family (EGFR,Her-2/neu, Her3 and Her4) is dependent upon the formation of homodimers,heterodimers or heterogenous multimers of these receptors triggered bythe binding of ligand. Bispecific scFv antibody molecules that engagemultiple epitope pairs of these receptor proteins have been generated asdescribed hereinbelow for use in preventing formation of these signalingcomplexes in cancerous tumor cells.

I. Materials and Methods:

The following materials and methods are provided to facilitate thepractice of the present invention:

A. Cloning:

All of the genes coding for single chain Fv (scFv) antibody moleculesspecific for the different members of the EGFR family (EGFR, HER2/neu,HER3, HER4) were obtained from Dr. Jim Marks (University of CaliforniaSan Francisco). The scFv genes were isolated from large naïve human scFvlibraries. The scFv genes specific for the EGFR proteins were isolatedby selection against the extracellular domains of these proteins. All ofthe scFv genes were provided as inserts in a pUC119myc/his vector,between the NcoI and NotI restriction sites. Sequences for these armsare set forth in Appendix A (SEQ ID NOS: 1-10, 19, and 21).

1. Construction of 20 Amino Acid Linker Molecule:

Proteolytic degradation of the bs-scFv antibody molecules in circulationmay limit their effectiveness. Thus, a novel 20 amino acid linker thatwas devoid of all known proteolytic sites was designed and synthesized.The amino acids employed in the construction of the linker were selectedto be primarily neutral (not charged, hydrophobic or hydrophilic) tofacilitate efficient transport of the protein into the bacterialperiplasmic space. The following two primers were synthesized whichencode the new linker molecule:

LW583 (5′-AAT TCA GGT GCT GGT ACT TCA GGT TCA GGT GCT TCA GGT GAA GGT TCA GGT TCA A- 3′, SEQ ID NO:  12); andLW584 (5′-AGC TTT GAA CCT GAA CCT TCA CCT GAA GCACCT GAA CCT GAA GTA CCA GCA CCT G- 3′, SEQ ID NO: 13).

Hybridization of these oligonucleotides formed a “sticky” ends linkerwith EcoRI and HindIII digested ends. This product was inserted into thepET20b(+) vector previously digested with EcoRI and HindIII. Plasmid DNAwas generated from transformed DH5αE. coli using a commerciallyavailable kit for DNA plasmid isolation and purification (Qiagen orGibco BRL Co.) and was subsequently named “pET20b(+)/Linker”. The linkermolecule is encoded by the following nucleic acid sequence: 5′-AAT TCAGGT GCT GGT ACT TCA GGT TCA GGT GCT TCA GGT GAA GGT TCA GGT TCA AAGCTA→3 (SEQ ID NO: 14), and the resulting linker molecule has thefollowing amino acid sequence: NSG AGT SGS GAS GEG SGS KL (SEQ ID NO:11).

2. Cloning Anti-HER3 Gene into pET20b(+)/Linker Vector:

The gene coding for the anti-HER3 scFv antibody molecule, A5, wasamplified from the A5-pUC119myc/his plasmid with the following twoprimers: LW687 (5′-CGA CCA TGG CCC AGG TGC AGC TGG TGC AG-3′, SEQ ID NO:15); and LW688 (5′-CGA ATT CAC CTA GGA CGG TCA GCT TGG-3′, SEQ ID NO:16).

The amplified product and vector, pET20b(+)/Linker, were both digestedwith NcoI and EcoRI enzymes, ligated and transformed into competentDH5αE. coli for plasmid DNA production. Selected enzymes directed the A5gene upstream from the linker. The new plasmid, called“pET20b(+)A5/Linker”, was then isolated and purified.

3. Cloning Anti HER2/Neu Gene into pET20b(+)A5/Linker Vector:

The gene coding for the anti-HER2/neu scFv antibody molecule, ML3.9, wasamplified from the ML3.9-pUC119myc/his plasmid using the following twoprimers: LW697 (5′-GGG AAG CTT CAG GTG CAG CTG GTG CAG TCT GG-3′, SEQ IDNO: 17); and LW698 (5′-GGG CTC GAG ACC TAG GAC GGT CAG CTT GGT TCC-3′,SEQ ID NO: 18)

The PCR amplified product and plasmid DNA, pET20b(+)A5/Linker, weredigested with HindIII and XhoI restriction enzymes, ligated andtransformed into competent DH5αE. coli for production of the new plasmidDNA, pET20b(+)A5/Linker/ML3.9. Selected enzymes directed the ML3.9 genedownstream from the linker sequence. The new plasmid, calledApET20b(+)A5/Linker/ML3.9″, was then isolated and purified.

4. Cloning of the A5/Linker/ML3.9 Gene into pUC119/myc/his Vector:

The nucleic acid molecule encoding the bs-scFv product from pET20b(+)was cloned into a pUC119myc/his vector. A (histidine)₆ tag and one“stop” codon, which are part of the pET vector, were amplified togetherwith the A5/Linker/ML3.9 nucleic acid construct. PCR amplification wasperformed using the following two primers: LW687 (5′-CGA CCA TGG CCC AGGTGC AGC TGG TGC AG-3′, SEQ ID NO: 5); and LW686 (5′-GAT ATA ATG CGG CCGCTC AGT GGT GGT GGT GGT G-3′, SEQ ID NO: 9)

Digestion of the pUC119myc/his vector and amplified product with NcoIand NotI enzymes was followed by a ligation step and transformation ofthe DH5αE. coli. The resulting plasmid DNA, called “pUC/ALM”, was thenpurified and isolated (FIG. 1).

B. Transformation of the Expression Clone, TG1:

pUC/ALM was transformed into E. coli strain, TG1, and the clonesproducing the A5/Linker/ML3.9 bs-scFv antibody molecules were isolatedas follows. The bs-scFv molecules were dialyzed overnight, purified byimmobilized metal affinity chromatography using Ni-NTA resin (Qiagen),followed by size-exclusion chromatography on an HPLC system using aSuperdex-75 column (Pharmacia).

II. Results:

As a proof of concept, two different bs-scFv antibody molecules werecreated. The first, named “ALM”, was composed of the AS scFv and theML3.9 scFv which specifically binds to both HER3 and HER2/neu,respectively. The second bs-scFv antibody molecule, named “ALF”, wascomposed of two distinct scFv molecules, AS and F4, both with aspecificity for HER3. Both bs-scFv antibody molecules were cloned andexpressed from E. coli.

ALM was evaluated in a series of in vitro and in vivo assays. Itsability to simultaneously bind to both HER3 and HER2/neu, individuallyand simultaneously, was demonstrated by surface plasmon resonance on aBIAcore instrument (FIGS. 2, 3 and 4). In vitro, incubation of ALM withhuman BT-474 breast cancer cells overexpressing both HER3 and HER2/neulead to reduced cell surface expression of HER2/neu and HER 3 (FIG. 5),decreased proliferation in MTT assays (FIG. 6), reduced survival in aclonogenicity assay (FIG. 7) and increased phosphorylation followed bymarked dephosphorylation of AKT2 (FIG. 8), an important protein in theapoptotic pathway. These effects were comparable (MTT assay) or greater(dephosphorylation of AKT2) than those observed using Herceptin⁷ (datanot shown).

In vivo, radioiodinated ALM exhibited enhanced specific tumor targetingwithin 24 hours after administration to immunodeficient mice bearings.c. human BT-474 tumor xenografts (FIG. 9).

These results demonstrate the utility of the bs-scFv antibody moleculesof the invention for the treatment of tumor cells that overexpress EGFRproteins. The novel bs-scFv antibody molecules can be used alone or incombination with existing chemotherapeutic methods to treat a variety ofcancers including, but not limited to breast, colon, ovarian,endometrial, gastric, pancreatic, prostate and salivary gland cancers.

Example 2 Combined Chemotherapeutic Approaches

HER2/neu is a compelling target for combined chemotherapy approaches asit is overexpressed in a variety of tumors and its overexpression hasbeen correlated with a poor prognosis. While HER2/neu lacks a ligandthat can trigger signaling through its tyrosine kinase domain, whenoverexpressed at high concentrations, HER2/neu can spontaneously formhomodimers (Yarden and Sliwkowski (2001) Nature Reviews, Molecular CellBiology 2: 127-137). HER3 is in many ways the opposite of HER2/neu. Itactively binds to ligand but lacks a functional tyrosine kinase domain,thus requiring heterodimerization with HER2/neu for signaling. In fact,this combination is believed by many to be the most potent of thesignaling complexes formed by the members of the EGFR family (Lohrischand Piccart (2001) Sem. Oncology (28) Suppl 18: 3-11).

Many chemotherapeutic agents lead to damage that in a normal cell willtrigger apoptosis. However, some tumor cells have aberrant signalingthat interferes with the normal apoptosis signaling pathway. Thephosphorylation of AKT2 in HER2/neu overexpressing tumor cells leads toan anti-apoptotic cascade that could interfere with the antitumoreffects of chemotherapeutic or biological agents (Zhou et al. (2000) J.Biol. Chem., 275: 8027-8031). Thus, targeting HER2/neu with bs-scFvantibody molecules in combination with existing chemotherapeutictreatments will be more effective in killing the tumor cells thanchemotherapy alone.

Example 3 In Vivo Efficacy of ²¹¹At-Labeled Bispecific scFv

An in vivo study was conducted to evaluate the efficacy of ²¹¹At labeledbispecific scFv against tumors. The bispecific antibody as labeled using²¹¹At-SAPS chelate (N-(4-[²¹¹At] astatophenethyl)succinimate) (see,e.g., FIG. 10).

Four days before injection of BT474 breast cancer cells, mice wereimplanted with a β-estradiol tablet. On day zero, the mice were injectedwith 5×10⁶ BT474 breast cancer cells. On day 14, the first therapeuticdose of ²¹¹AT conjugated bispecific antibody (ALM) was administered i.p.at a high dose of 80 μg and at a low dose of 10 μg. Subsequenttherapeutic doses were administered on day 16 and on day 18. Tumorvolume was then tracked as shown in FIGS. 11A through 11C.

Tumor volume was generally lower in the treated animals (FIGS. 11B and11C) as compared to the untreated control (FIG. 11A).

Example 4 Cancer Imaging

FIG. 12 shows a PET-CT image of two mice using Iodine-124 labeled ALMbispecific single-chain Fv. The mice were injected i.v. with 50microCuries (50 micrograms) of labeled ALM and were imaged 48 hourslater.

This should illustrates the efficacy of the bispecific antibodies ofthis invention for the detection of cancer.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the purview of this application and the scope ofthe appended claims. All publications, patents, and patent applicationscited herein and accompanying appendices are hereby incorporated byreference in their entirety for all purposes.

What is claimed is:
 1. A method for treating a cancer that overexpressesHER2 and/or HER3 proteins, said method comprising administering to apatient in need thereof a therapeutically effective amount of abispecific antibody molecule comprising a first scFv antibody that bindsHER2/neu and a second scFv antibody that binds HER3, wherein the firstscFv antibody binds an epitope of HER2/neu specifically bound byantibody C6-B1D2 and the second scFv antibody binds an epitope of HER3specifically bound by antibody HER3.H3.
 2. The method of claim 1,wherein the cancer overexpresses HER2.
 3. The method of claim 2, whereinthe cancer overexpresses HER2 and HER3.
 4. The method of claim 1,wherein the molecule is a single contiguous polypeptide.
 5. The methodof claim 4, wherein the cancer overexpresses HER2.
 6. The method ofclaim 5, wherein the cancer overexpresses HER2 and HER3.
 7. The methodof claim 4, wherein the first antibody is joined to the second antibodyby a linker.
 8. The method of claim 7, wherein the cancer overexpressesHER2.
 9. The method of claim 8, wherein the cancer overexpresses HER2and HER3.
 10. The method of claim 7, wherein the linker is a peptidelinker.
 11. The method of claim 10 wherein the cancer overexpressesHER2.
 12. The method of claim 11, wherein the cancer overexpresses HER2and HER3.
 13. The method of claim 1, wherein the specific binding to anepitope of HER2/neu is determined by ability to compete with the C6-B1D2for binding to HER2/neu and specific binding to an epitope of HER3 isdetermined by ability to compete with HER3.H3 for binding to HER3. 14.The method of claim 2, wherein the specific binding to an epitope ofHER2/neu is determined by ability to compete with the C6-B1D2 forbinding to HER2/neu and specific binding to an epitope of HER3 isdetermined by ability to compete with HER3.H3 for binding to HER3. 15.The method of claim 4, wherein the specific binding to an epitope ofHER2/neu is determined by ability to compete with the C6-B1D2 forbinding to HER2/neu and specific binding to an epitope of HER3 isdetermined by ability to compete with HER3.H3 for binding to HER3. 16.The method of claim 5, wherein the specific binding to an epitope ofHER2/neu is determined by ability to compete with the C6-B1D2 forbinding to HER2/neu and specific binding to an epitope of HER3 isdetermined by ability to compete with HER3.H3 for binding to HER3. 17.The method of claim 7, wherein the specific binding to an epitope ofHER2/neu is determined by ability to compete with the C6-B1D2 forbinding to HER2/neu and specific binding to an epitope of HER3 isdetermined by ability to compete with HER3.H3 for binding to HER3. 18.The method of claim 8, wherein the specific binding to an epitope ofHER2/neu is determined by ability to compete with the C6-B1D2 forbinding to HER2/neu and specific binding to an epitope of HER3 isdetermined by ability to compete with HER3.H3 for binding to HER3. 19.The method of claim 10, wherein the specific binding to an epitope ofHER2/neu is determined by ability to compete with the C6-B1D2 forbinding to HER2/neu and specific binding to an epitope of HER3 isdetermined by ability to compete with HER3.H3 for binding to HER3. 20.The method of claim 11, wherein the specific binding to an epitope ofHER2/neu is determined by ability to compete with the C6-B1D2 forbinding to HER2/neu and specific binding to an epitope of HER3 isdetermined by ability to compete with HER3.H3 for binding to HER3. 21.The method of claim 12, wherein the specific binding to an epitope ofHER2/neu is determined by ability to compete with the C6-B1D2 forbinding to HER2/neu and specific binding to an epitope of HER3 isdetermined by ability to compete with HER3.H3 for binding to HER3.